EA039788B1 - [1,2,4]TRIAZOLO[1,5-a]PYRIMIDINE DERIVATIVES AS PDE2 INHIBITORS - Google Patents

Abstract

The invention relates to novel [1,2,4]triazolo[1,5-a]pyrimidinyl derivatives characterized by formula (I) as inhibitors of phosphodiesterase 2 (PDE2). The invention is also directed to pharmaceutical compositions comprising compounds, to processes for preparing such compounds and compositions, and to the use of such compounds and compositions for the prevention and treatment of disorders in which PDE2 is involved, such as neurological and psychiatric disorders.

Description

The field of technology to which the invention belongs

The present invention relates to novel [1,2,4]triazolo[1,5-a]pyrimidinyl derivatives as phosphodiesterase 2 (PDE2) inhibitors. The present invention is also directed to pharmaceutical compositions containing the compounds, to methods for preparing such compounds and compositions, and to the use of such compounds and compositions for the prevention and treatment of disorders in which PDE2 is involved, such as neurological and psychiatric disorders.

Background of the invention

Phosphodiesterases (PDEs) are a family of enzymes encoded by 21 genes and subdivided into 11 distinct families according to structural and functional properties. These enzymes mediate metabolic inactivation of the widely distributed intracellular second messengers, cyclic 3',5'-adenosine monophosphate (cAMP) and cyclic 3',5' guanosine monophosphate (cGMP). These two messengers regulate a wide variety of biological processes, including the production and action of proinflammatory mediators, ion channel function, muscle contraction, commitment, differentiation, apoptosis, lipogenesis, glycogenolysis, and gluconeogenesis. They do this by activating protein kinase A (PKA) and protein kinase G (PKG), which in turn phosphorylate a wide variety of substrates, including transcription factors and ion channels that regulate numerous physiological responses. In the case of neurons, activation of cAMP- and cGMP-dependent kinases and subsequent phosphorylation of proteins involved in the rapid regulation of synaptic transmission, as well as in differentiation and survival of neurons, are envisaged. Intracellular concentrations of cAMP and cGMP are strictly regulated by the rate of biosynthesis by cyclases and the rate of cleavage by PDE. PDEs are hydrolases that inactivate cAMP and cGMP via catalytic hydrolysis of the 3'-ester bond to form an inactive 5'-monophosphate (Scheme A).

Scheme A:

cAMP X = NH ₂ , Y = H cGMP X = O, Y = NH ₂

Based on substrate specificity, the PDE families can be divided into three groups: i) cAMP-specific PDEs, which include PDE4, 7 and 8; ii) cGMP-selective enzymes PDE5, 6 and 9; and iii) PDEs acting on two substrates, PDE1, 2 and 3, and PDE10 and 11.

Moreover, PDE is characterized by differential expression throughout the body, including in the central nervous system. As a consequence, different PDE isoenzymes may have different physiological functions. Compounds that selectively inhibit PDE families or isoenzymes may exhibit particular therapeutic activity, fewer side effects, or both.

Phosphodiesterase 2A (PDE2A) inactivates intracellular signaling mechanisms that depend on cAMP and cGMP mediated cyclic nucleotide signaling by cleaving them (by hydrolyzing the biologically important second messengers cAMP and cGMP to produce non-signaling AMP and GMP respectively). Such signaling pathways are known to play a role in the regulation of genes involved in the induction of synaptic plasticity. Pharmacological inhibition of PDE2 thus causes increased levels of synaptic plasticity (the correlate underlying learning and memory formation), indicating that modulation of PDE2A may be a target for alleviating the cognitive impairments seen in individuals suffering from such disorders, such as schizophrenia, Alzheimer's disease, Parkinson's disease and other CNS disorders associated with cognitive dysfunction.

Phosphodiesterase 2A (PDE2A) is expressed in greater amounts in the brain than in peripheral tissues. The high level of PDE2 expression in the limbic system (isocortex, hippocampus, amygdala, epithalamic leash, nucleus basalis) indicates that PDE2 can modulate interneuronal signaling associated with emotion, perception, attention, learning, and memory. In addition, PDE2 is expressed in the nucleus accumbens, olfactory bulb, olfactory tubercle, and amygdala, suggesting that PDE2 may also be involved in anxiety and depression (see, e.g., Lakics, V. et al. (2010) Quanti - 1 039788 tative comparison of phosphodiesterase mRNA distribution in human brain and peripheral tissues Neuropharmacol 59, 367-374).

In addition, PDE2 inhibitors have been shown to be effective in attenuating oxidative stress-induced anxiety, suggesting their use in the treatment of anxiety in neuropsychiatric and neurodegenerative disorders that involve oxidative stress, such as Alzheimer's disease, Parkinson's disease, and disseminated sclerosis.

PDE2 inhibitors have been shown to enhance long-term potentiation of synaptic transmission and improve recall and memory consolidation in tests of object recognition and social orientation in rats. Moreover, PDE2 inhibitors have been shown to reverse MK-801-induced short-term memory impairment in the T-maze in mice. PDE2 inhibitors have also been shown to be active in the forced swim test and in light/dark chamber models; and also show anxiolytic-like effects in the elevated plus maze, holed platform, and open field tests and prevent stress-induced changes in apoptosis and behavior.

Thus, PDE2 inhibitors can be used in the treatment of memory impairment, cognitive impairment, anxiety, bipolar disorder and depression.

WO 2015/164508 (Dart Neuroscience, LLC) discloses substituted [1,2,4]triazolo[1,5a]pyrimidinyl compounds as PDE2 inhibitors. WO 2017/076900 (Janssen Pharmaceutica NV) discloses [(3S,4R)-3-[5-(difluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl]-4 -methyl-1-piperidyl]-(2,6-dimethyl-4-pyridyl)methanone.

There is still a need for PDE2 inhibitor compounds with an advantageous combination of properties, eg improved potency, better selectivity for PDE3 and/or PDE10 and/or better chemical or metabolic stability.

Brief description of the invention

The aim of the present invention is to provide new PDE2 inhibitors that may be potentially useful in the treatment of diseases associated with the activity of the PDE2 enzyme.

Thus, the present invention is directed to compounds of formula (I)

and their stereoisomeric forms, where

R ^a is selected from the group consisting of H, CH ₃ , CN and CHF ₂ ;

R ^B is a radical selected from the group consisting of (a) and (c)

(a) , (c), where

R1 is H, F or CH3;

R ² is H, methyl or n-butyl;

with the proviso that if R ² is H, then R 1 is F or CH3;

R ³ is Ar, Het or Ar-C _2-4 alkenyl; where

Ar is phenyl or naphthyl, each of which is optionally substituted with 1, 2 or 3 substituents, each independently selected from the group consisting of halogen; CN;

NR ^2A R ^2B where R ^2A and R ^2B are each independently selected from H and CH ₃ ; OH; C _1-6 alkyl optionally substituted with 1, 2 or 3 independently selected halogen substituents;

C _1-6 alkyl substituted with CN; C _3-6 cycloalkyl;

C _1-6 alkyloxy optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; and pyrazolyl;

Het is (i) a 5-membered heteroaryl selected from the group consisting of 1H-pyrrole; thienyl; furanyl; W-pyrazolyl; W-imidazolyl; 1,2-oxazolyl; 1,3-oxazolyl and thiazolyl; each of which may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halogen; C _1-4 alkyl optionally substituted with 1, 2, or 3 independently selected

- 2,039,788 substituents representing a halogen atom; NR ^3A R ^3B , where R ^3A and R ^3B are each independently selected from H and CH ₃ ; and furan-2-yl; or (ii) a 6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl; each of which may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halogen; OH; CN; NR ^4A R ^4B , where R ^4A and R ^4B are each independently selected from H and CH ₃ ; C ₁ _ ₄ αalkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; C ₁ _ ₄ alkyl substituted with OH; C ₃ _ ₆ cycloalkyl; C ₃ _ ₆ cycloalkyloxy; C ₁ _ ₄ αkyloxy; C ₁ _ ₄ alkyloxyC ₁ _ ₄ alkyl; or (iii) an 8-10 membered bicyclic partially unsaturated heterocyclyl selected from the group consisting of 2,3-dihydro-1-benzofuranyl; 2H-chromenyl; 3,4-dihydro-2H-chromenyl; 2,3-dihydro-Shindolyl optionally substituted in position 1 with 1-acetyl; 2,2-difluoro-1,3-benzodioxolyl; 1,3benzodioxolyl optionally substituted with a methyl substituent; 3,4-dihydro2H-1,4-benzoxazinyl optionally substituted with C ₁ _ ₄ alkyl; 5,6,7,8-tetrahydroimidazo[1,2a]pyridinyl; 5,6,7,8-tetrahydroquinolinyl optionally substituted with a halogen substituent; and 2,3-dihydropyrazolo[5,1-b][1,3]oxazolyl; or (iv) a 9-10 membered bicyclic heteroaryl selected from the group consisting of 1-benzofuranyl; 1-benzothiophenyl; 1H-indolyl; 1,3-benzoxazolyl; 1,3-benzothiazolyl; indolizinyl; 1Hbenzimidazolyl; imidazo[1,2-a]pyridinyl; pyrazolo[1,5-a]pyridinyl; 1H-thieno[2,3-e]pyrazolyl; imidazo[2,1-b]thiazolyl; pyrrolo[2,3-e]pyridinyl; thieno[3,2-b]pyridinyl; quinolinyl; isoquinolinyl; quinoxalinyl; 1,8-naphthyridinyl and 1,6-naphthyridinyl; each of which may be optionally substituted with 1 or 2 substituents each independently selected from the group consisting of halogen; OH; NR ^5A R ^5B , where R ^5A and R ^5B are each independently selected from H and CH ₃ ; C ₁ _ ₄ αalkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; and C ₁ _ ₄ alkyloxy;

provided that the connection is not

or its N-oxide, or pharmaceutically acceptable salt, and N-oxides, and their pharmaceutically acceptable salts. An illustration of the present invention is a pharmaceutical composition containing a pharmaceutically acceptable carrier and a compound of formula (I) described herein, or a pharmaceutically acceptable salt or solvate thereof. An illustration of the present invention is a pharmaceutical composition obtained by mixing a compound of formula (I) described herein, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier. An illustration of the present invention is a process for preparing a pharmaceutical composition comprising mixing a compound of formula (I) described herein, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

An example of the present invention are methods of treating a disorder mediated by a PDE2 enzyme, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) described herein, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical compositions described herein. .

An example of the present invention is a method of treating a disorder selected from the group consisting of neurological and psychiatric disorders, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) described herein, or a pharmaceutically acceptable salt or solvate thereof, or pharmaceutical compositions described in this document.

An example of the present invention is a method of treating a disorder selected from the group of neurological and psychiatric disorders selected from psychotic disorders and conditions; anxiety disorders; movement disorders; drug addiction; affective disorders; neurodegenerative disorders; disorders or conditions including attention deficit disorder and/or cognitive impairment as a symptom; stroke and autistic disorders, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) described herein, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically acceptable salt or solvate thereof, or pharmaceutical compositions described in this document.

An example of the present invention is a method of treating a disorder selected from the group consisting of neurological and psychiatric disorders, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) described herein, or a pharmaceutically acceptable salt or solvate thereof, or pharmaceutical compositions described in this document.

An example of the present invention is a method of treating a disorder selected from the group of neurological and psychiatric disorders selected from psychotic disorders and conditions; anxiety disorders; movement disorders; drug addiction; affective disorders; neurodegenerative disorders; disorders or conditions including attention deficit disorder and/or cognitive impairment as a symptom; disorders associated with memorization and memory consolidation; stroke and autistic disorders, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I), or a salt or solvate thereof, or the pharmaceutical compositions described herein.

Also an example of the present invention is a compound of formula (I), or a salt or solvate thereof, or a pharmaceutical composition described herein, for use as a drug.

A further example of the present invention is a compound of formula (I), or a salt or solvate thereof, or a pharmaceutical composition according to the present invention, for use in the treatment, prevention, amelioration, control, or risk reduction of various neurological and psychiatric disorders associated with phosphodiesterase 2 function in a mammal, including humans, the treatment or prevention of which depends on or is facilitated by inhibition of phosphodiesterase 2.

An example of the present invention is a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof according to the present invention, or a pharmaceutical composition according to the present invention, for use in the treatment, prevention, amelioration, control, or risk reduction of various disorders selected from psychotic disorders and conditions; anxiety disorders; movement disorders; drug addiction; affective disorders; neurodegenerative disorders; disorders or conditions including attention deficit disorder and/or cognitive impairment as a symptom; disorders associated with memorization and memory consolidation; stroke and autistic disorder.

An example of the present invention is a method of treating a disorder selected from the group consisting of Alzheimer's disease, mild cognitive impairment, senile dementia, dementia, Lewy body dementia, Down syndrome, stroke-associated dementia, Parkinson's disease-associated dementia, and dementia, associated with beta-amyloid deposition, preferably Alzheimer's disease, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical compositions described herein.

Another example of the present invention is a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described herein, for use in the treatment of: (a) Alzheimer's disease, (b) mild cognitive impairment, (c) senile dementia, (d) dementia, (e) Lewy body dementia, (f) Down syndrome, (g) stroke-associated dementia, (h) Parkinson's disease-associated dementia, (i) beta-amyloid deposition-associated dementia, (j) depressive disorders and (k) anxiety disorders in a subject in need thereof.

Description of graphic materials

In FIG. 1 shows the effect of compound 110 on induction by weak HFS long-term potentiation (LTP) at the mossy fiber synapse.

In FIG. 2 shows the experimental plan for theta-pulse stimulation.

In FIG. 3 shows the effect of compound 70 on induction by weak HFS long-term potentiation (LTP) at the mossy fiber synapse.

In FIG. 4 shows the effect of compound 25a on induction by weak HFS long-term potentiation (LTP) at the mossy fiber synapse.

In FIG. 5 shows the effect of compound 220 on induction by weak HFS long-term potentiation (LTP) at the mossy fiber synapse.

In FIG. 6-15 (compounds 220, 110, 93, 32, 70, 25a, 281, 295, 356 and 335, respectively) show the measurement of cGMP levels in CSF in Marshall Beagle dogs. In FIG. 6-15 various doses of test compounds are presented as follows: 0 mg/kg; • 0.5 mg/kg; ♦ 0.75 mg/kg; ▲ 1 mg/kg; ▼ 1.25 mg/kg; X 1.5 mg/kg; V 2.5 mg/kg.

In FIG. 16 shows the transient effects of Compound 110 on in vivo synaptic function at the Schaffer (SC)-CA1 collateral synapse. In FIG. 16a shows a schematic representation of the location of a bipolar stimulating electrode in the Schaffer collateral tract and a monopolar recording electrode in the stratum radiatum region of the Cornu Ammonis CA1 of the hippocampus; in fig. 16b shows a summary of the I/O curves obtained by applying increasing intensity stimuli and measuring the initial slopes of the resulting fEPSP, which indicate no difference in the underlying excitability of the SC-CA1 pathway; in fig. 16c shows response morphology parameters at baseline, 30 min after treatment, and 30 min after the tetanic HFS protocol. Of note is the similarity in magnitude of fEPSP responses during recording of baseline levels between treatment groups, which increased above baseline, and the conditions of use of the vehicle medium in the group treated with Compound 110; in fig. 16d shows the time dependence of the fEPSP slope at 30 min post baseline, 30 min after compound 110 administration, and using the tetanic HFS protocol and recording the fEPSP slope for another 2 h. Note that compound 110 at dose 20 and 40 mg/kg increased basal synaptic transmission before the tetanic HFS protocol and increased the slope of fEPSP over 2 h after the HFS protocol (inserted graph); in fig. 16e shows that when fEPSP was averaged over 30 min periods, Compound 110 (40 mg/kg) provided an increase in the amount of synaptic transmission and the difference from the vehicle treated group remained significant. Values are expressed as a percentage of pre-HFS values and results are presented as means±SEM. For group comparisons, tests with one-way analysis of variance (ANOVA) and post hoc analysis with the least significant difference (LSD) were used.

In FIG. 16b and c diagrams have the following designations:

carrier (n=10); compound 110 (mg/kg) (20) n=9; compound 110 (mg/kg) (40) n=8.

In FIG. 16d and 16e diagrams have the following designations:

carrier (n=10); th compound 110 (mg/kg), (20) n=9; compound 110 (mg/kg) (40) n=8.

Detailed Description of the Invention Definition

C ₁ .4αalkyl, used herein alone or as part of another group, means a saturated, straight or branched hydrocarbon radical containing 1, 2, 3 or 4 carbon atoms, such as methyl, ethyl, 1-propyl, 1-methyl, butyl, 1-methylpropyl, 2-methyl-1-propyl, 1,1-dimethylethyl, and the like. Term C ₁ . ₆ alkyl, as used herein as a group or part of a group, means a straight or branched saturated hydrocarbon radical containing from 1 to 6 carbon atoms, such as the groups defined for C ₁ .4 alkyl, and n-pentyl, n- hexyl, 2-methylbutyl, and the like. C ₁ .4alkyloxy will mean an ether radical, where O^alkyl is as defined herein. _C1 . ₆ αkyloxy will mean an ether radical, where C ₁ . ₆ alkyl is as defined herein. Halogen will denote fluorine, chlorine or bromine. _C3 . ₆ cycloalkyl will mean cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. _C3 . ₆ cycloalkyloxy will mean an ether radical where C ₃ . ₆ cycloalkyl is as defined herein.

The term substituted in all uses in the present invention is intended to mean, unless otherwise indicated or clearly implied by the context, that one or more hydrogen atoms, preferably 1 to 3 hydrogen atoms, or 1 to 2 hydrogen atoms, or 1 a hydrogen atom, at an atom or radical denoted using the expression substituted, is replaced by a member selected from the indicated group, provided that the normal valence is not exceeded, and that the substitution results in a chemically stable compound, i.e. a compound that is sufficiently stable to withstand isolation from the reaction mixture to a suitable degree of purity and formulation into a therapeutic agent.

The N-oxide forms of the compounds of formula (I) are intended to include those compounds of formula (I) in which one or more nitrogen atoms are oxidized to the so-called N oxide, in particular those N-oxides in which the nitrogen atom in the pyridinyl radical is oxidized . Noxides can be obtained according to procedures known to the person skilled in the art. The N-oxidation reaction can generally be carried out by reacting the starting material of formula (I) with an appropriate organic or inorganic peroxide. Suitable inorganic peroxides include, for example, hydrogen peroxide, peroxides of alkali and alkaline earth metals, eg sodium peroxide, potassium peroxide; suitable organic peroxides may include peroxyacids such as, for example, benzenecarboperoxyacids or halogenated benzenecarboperoxyacids, such as 3-chloroperoxybenzoic acid (or 3-chloroperbenzoic acid), peroxyalkanoic acids, such as peroxyacetic acid, alkyl hydroperoxides, such as tert-butyl hydroperoxide. Suitable solvents are, for example, water, lower alkanols, eg ethanol and the like, hydrocarbons, eg toluene, ketones, eg 2-butanone, halogenated hydrocarbons, eg dichloromethane, and mixtures of such solvents.

Thus, in a specific embodiment, the present invention relates to a compound of formula (I) wherein R ³ is Het and Het is the oxide of the optionally substituted pyridyl radical described herein, i.e. an optionally substituted radical in the form of pyridinium oxide.

- 5 039788

The term subject as used herein refers to an animal, preferably a mammal, most preferably a human, that is or has been the subject of treatment, observation, or experiment.

The term therapeutically effective amount, as used herein, means that amount of an active compound or pharmaceutical agent that elicits a biological or medical response in a tissue system, animal, or human that a researcher, veterinarian, physician, or other clinician seeks to obtain, which includes the relief of symptoms. the disease or disorder being treated.

As used herein, the term composition is intended to cover a product containing certain ingredients in certain amounts, as well as any product that is obtained directly or indirectly from combinations of certain ingredients in certain amounts.

Hereinafter and hereinafter, the term compound of formula (I) is to be understood as including its addition salts, solvates and stereoisomers.

The terms stereoisomers or stereochemically isomeric forms are used interchangeably throughout this document.

The present invention includes all stereoisomers of a compound of formula (I), either as a pure stereoisomer or as a mixture of two or more stereoisomers.

Enantiomers are stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of enantiomer pairs is a racemate or racemic mixture. Diastereomers (or diastereoisomers) are stereoisomers that are not enantiomers, i.e. they are not related as mirror images. Therefore, the present invention includes enantiomers, diastereomers, racemates.

In the compounds of the present invention, bonds shown as a wedge of parallel lines ( ¹ 1 ) are bonds projected below the plane of the graphic, while bonds shown as a thick wedge (--) are bonds projected above the plane of the graphic. Images.

The absolute configuration is in accordance with the Cahn-Ingold-Prelog system. The configuration at an asymmetric atom is indicated as R or as S. Separated compounds whose absolute configuration is unknown may be indicated as (+) or (-), depending on the direction in which they rotate plane polarized light.

If a specific stereoisomer is indicated, this means that the specified stereoisomer contains practically no other isomers, i.e. associated with less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, in particular less than 2% and most preferably less than 1% of other isomers. Thus, if a compound of formula (I) is indicated, for example, as (R), this means that the compound contains essentially no (S)-isomer.

In addition, some crystalline forms of the compounds of the present invention may exist as polymorphs and are intended to be included as such in the present invention.

In addition, some of the compounds of the present invention may form solvates with water (ie, hydrates) or common organic solvents, and such solvates are also intended to be within the scope of the present invention.

With regard to use in medicine, the salts of the compounds of the present invention refer to non-toxic pharmaceutically acceptable salts. However, other salts may be useful in the preparation of the compounds of the present invention or their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds include acid addition salts which can be formed, for example, by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. In addition, when the compounds of the present invention have an acid moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, for example sodium or potassium salts; alkaline earth metal salts, for example calcium or magnesium salts; and salts formed with suitable organic ligands, for example quaternary ammonium salts.

Typical acids that can be used in the preparation of pharmaceutically acceptable salts include, without limitation, the following: acetic acid, 2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, Laspartic acid, benzenesulfonic acid, benzoic acid, 4 -acetamidobenzoic acid, (+)-camphoric acid, camphorsulfonic acid, caproic acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, ethane-1,2disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucuronic acid, L-glutamic acid, beta-oxoglutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, (+ )-b-lactic acid, (±)-DL-lactic acid, lactobionic acid oto, maleic acid, (-)H-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid , nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid , (+)-b-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoromethylsulfonic acid and undecylenic acid. Representative bases that can be used in the preparation of pharmaceutically acceptable salts include, without limitation, the following: ammonia, L-arginine, benetamine, benzathine, calcium hydroxide, choline, dimethylethanolamine, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N -methylglucamine, hydrabamine, III-imidazole, L-lysine, magnesium hydroxide, 4-(2hydroxyethyl)morpholine, piperazine, potassium hydroxide, 1-(2-hydroxyethyl)pyrrolidine, secondary amine, sodium hydroxide, triethanolamine, tromethamine and zinc hydroxide.

The names of the compounds of the present invention were compiled according to the nomenclature rules agreed upon by the Chemical Abstracts Service (CAS) using software from Advanced Chemical Development, Inc. (ACD/Name: product version 10.01, build 15494, December 1, 2006; or ACD/ChemSketch: product version 12.5, build 47877, April 20, 2011) or according to the nomenclature rules agreed upon by the International Union of Pure and Applied Chemistry (IUPAC), using software from Advanced Chemical Development, Inc. (ACD/Name: product version 10.01.0.14105, October 2006). In the case of tautomeric forms, the name of the depicted tautomeric form of the structure was made. Another not shown tautomeric form is also included in the scope of the present invention.

The present invention is directed to the compounds of formula (I) as defined herein above and their pharmaceutically acceptable salts and solvates.

In one embodiment, the present invention is directed to compounds of formula (I), k. "A

R ^B (I) , and their stereoisomeric forms, where

R ^a is selected from the group consisting of H, CH3, CN and CHF2; R ^B is a radical selected from the group consisting of (a), (b) and (c):

where

R ¹ is H, F or CH3;

R ² is H or C ₁ . ₄ alkyl, in particular methyl or n-butyl; with the proviso that if R ² is H, then R ¹ is F or CH3;

R ³ is Ar or Het; where

Ar is phenyl optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halo; CN; OH; _C1 . ₆ alkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; _C1 . ₆ αkyl substituted with CN; _C3 . ₆ cycloalkyl and C ₁ . ₆ alkyloxy optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom;

Het is (i) a 5-membered heteroaryl selected from the group consisting of 1Hpyrrolyl; thienyl; furanyl; 1H-pyrazolyl; 1H-imidazolyl; 1,2-oxazolyl; 1,3-oxazolyl and thiazolyl; each of which may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halogen; _C1 . ₄ alkyl optionally substituted with 1, 2, or 3 independently selected substituents representing a halogen atom; NR ^3A R ^3B where R ^3A and R ^3B are each independently selected from H and CH3; and furan-2-yl; or

- 7 039788 (ii) 6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl; each of which may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halogen; OH; CN; NR ^4A R ^4B where R ^4A and R ^4b are each independently selected from H and CH3; _C1 . ₄ alkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; _C3 . ₆ cycloalkyl; C ₃ _ ₆ cycloalkyloxy; and C ₁ . ₄ alkyloxy optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; or (iii) an 8-10 membered bicyclic partially unsaturated heterocyclyl selected from the group consisting of 2,3-dihydro-1-benzofuranyl; 2H-chromenyl; 3,4-dihydro-2H-chromenyl; 2,3-dihydro-Shindolyl optionally substituted at the 1-position with C _1-4 alkyl, methylsulfonyl, 1-acetyl or fluoroacetyl; 2,2-difluoro-1,3-benzodioxolyl; 1,3-benzodioxolyl optionally substituted with a methyl substituent; 5,6,7,8-tetrahydroimidazo[1,2-a]pyridinyl; 5,6,7,8 tetrahydroquinolinyl optionally substituted with a halogen substituent; and 2,3-dihydropyrazolo[5,1-b][1,3]oxazolyl; or (iv) a 9-10 membered bicyclic heteroaryl selected from the group consisting of 1benzofuranyl; 1-benzothiophenyl; 1H-indolyl; 1,3-benzoxazolyl; 1,3-benzothiazolyl; indolizinyl; 1H-benzimidazolyl; imidazo[1,2-a]pyridinyl; pyrazolo[1,5-a]pyridinyl; 1H-thieno[2,3-c]pyrazolyl; thieno[3,2-b]pyridinyl; quinolinyl; 1,8-naphthyridinyl and 1,6-naphthyridinyl; each of which may be optionally substituted with 1 or 2 substituents each independently selected from the group consisting of halogen; OH; NR ^5A R ^5B , where R ^5A and R ^5B are each independently selected from H and CH ₃ ; _C1 . ₄ αalkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; and _C1 . ₄ alkyloxy optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom;

provided that the compound is not and their pharmaceutically acceptable salts and solvates.

In a particular embodiment, RA is CH3 or CHF ₂ ; and other variables are defined in this document.

In a particular embodiment, R ^B is selected from the group consisting of (a) and (c) and the remaining variables are defined herein.

In a particular embodiment, R ^A is CH3 or CHF2; R ^B is selected from the group consisting of (a) and (c), and the remaining variables are defined in this document.

In a specific embodiment, R ¹ is H and R ² is C ₁ . ₄ αalkyl, in particular methyl or n-butyl; and other variables are defined in this document.

In a particular embodiment, R ³ is Het and the remaining variables are defined herein.

In a particular embodiment, R ³ is (i) a 6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl; each of which may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halogen; OH; CN; NR ^4A R ^4B , where R ^4A and R ^4B are each independently selected from H and CH3;

_C1 . ₄ alkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; _C3 . ₆ cycloalkyl; _C3 . ₆ cycloalkyloxy and C ₁ . ₄ αalkyloxy optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; or (ii) a 9-10 membered bicyclic heteroaryl selected from the group consisting of 1benzofuranyl; 1-benzothiophenyl; 1H-indolyl; 1,3-benzoxazolyl; 1,3-benzothiazolyl; indolizinyl; 1H-benzimidazolyl; imidazo[1,2-a]pyridinyl; pyrazolo[1,5-a]pyridinyl;

1H-thieno[2,3-c]pyrazolyl; thieno[3,2-b]pyridinyl;

quinolinyl; 1,8-naphthyridinyl and 1,6-naphthyridinyl; each of which may be optionally substituted with 1 or 2 substituents, each independently selected from the group consisting of

- 8 039788 halogen; OH; NR ^5A R ^5B , where R ^5A and R ^5B are each independently selected from H and CH ₃ ;

C1-4alkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; and C _1-4 alkyloxy optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; and other variables are defined in this document.

In a further embodiment, R ³ is (i) a 6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidinyl, pyrazinyl, and pyridazinyl; each of which may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halogen; NR ^4A R ^4B , where R ^4A and R ^4B are each independently selected from H and CH3;

C _1-4 alkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; C _3-6 cycloalkyl; C _3-6 cycloalkyloxy and C _1-4 alkyloxy optionally substituted with 1, 2 or 3 independently selected halogen substituents;

or (ii) a 9-10 membered bicyclic heteroaryl selected from the group consisting of 1benzofuranyl; 1-benzothiophenyl; 1H-indolyl; 1,3-benzoxazolyl; 1,3-benzothiazolyl; indolizinyl; 1 H-benzimidazolyl; imidazo[1,2-a]pyridinyl; pyrazolo[1,5-a]pyridinyl;

H-thieno[2,3-c]pyrazolyl; thieno[3,2-b]pyridinyl;

quinolinyl; 1,8-naphthyridinyl; and 1,6-naphthyridinyl; each of which may be optionally substituted with 1 or 2 substituents each independently selected from the group consisting of halogen; NR ^5A R ^5B , where R ^5A and R ^5B are each independently selected from H and CH3;

C _1-4 alkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; and C _1-4 alkyloxy optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom;

and other variables are defined in this document.

In one embodiment, a compound of formula (I), as defined, is specifically a compound of formula (I-a) or (I-b), herein,

where all variables are defined in this document. More specifically, a compound of formula (I) as defined herein is characterized by formula (I-b1),

where all variables are defined in this document. In a specific embodiment, R ³ is a radical selected from the group consisting of (3-a), (3-b) and (3-c):

where

- 9 039788 each of R ^3a and R ^3b is independently selected from the group consisting of hydrogen; halogen; CN; NR ^4a R ^4b , where R ^4a and R ^4b are each independently selected from H and CH ₃ ; C _1-4 alkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; C _3-6 cycloalkyl and C _1-4 αalkyloxy optionally substituted with 1, 2 or 3 independently selected halogen substituents; and

R ^3c is selected from the group consisting of F, Cl, C1-3alkyl, cyclopropyl, C1-3alkyloxy, cyclopropyloxy and CF3; and each of R ^3d and R ^3e is independently selected from the group consisting of hydrogen, Cl, CN, C13 alkyl, cyclopropyl, C _1-3 alkyloxy, cyclopropyloxy, CHF ₂ , CF ₃ , OCHF ₂ and OCF ₃ ; provided that R ^3d and R ^3e are not hydrogen at the same time.

In particular, each of R ^3a and R ^3b is independently selected from the group consisting of hydrogen; chlorine; -NH2; C1-4alkyl; -CF3; cyclopropyl; -OCH3; -OSN(CH3)2; -OCHF2 and -OCF3.

More specifically, each of R ^3a and R ^3b is independently selected from the group consisting of chlorine; C1 ₄ alkyl; _-CF3 ; cyclopropyl; -OCH ₃ ; -OCH(CH ₃ ) ₂ ; -OCHF2 and _-OCF3 .

In particular, R ^3a is Cl or CH3, and R ^3b is selected from the group consisting of CH3, -OCH ₃ , -CF ₃ and cyclopropyl.

In a further embodiment, R ^3a is Cl or CH3 and R ^3b is CH3 or -OCH ₃ .

In particular, R ^3c is selected from the group consisting of F, CH3 and -OCH3; R ^3d is selected from the group consisting of hydrogen, CH3, cyclopropyl and -OCH3; and R ^3e is selected from the group consisting of Cl, CH3, cyclopropyl, -OCH3; -OCH(CH ₃ ) ₂ ; cyclopropyloxy; CHF2 and OCHF2. In a further embodiment, R ^3c is F.

In a further embodiment, the compound according to the present invention is a compound of formula (I-a) wherein R ² is C _1-4 αkyl, in particular methyl or n-butyl.

Getting connections

Experimental procedure 1:

Reaction scheme 1a:

Reaction scheme 1b.

A: combination to form an amide bond.

Final compounds where R ^B is a radical of formula (a) or formulas (b) or (c), herein referred to as compounds of formula (I-A) and formula (I-BC), where ---- is CH2 or CH2CH2, respectively, can be readily obtained by reacting with the appropriate carboxylic acid (III) using the following coupling procedures known in the art (reaction step A). This conversion can be easily carried out by treating the piperidine-type functional group in the intermediates of formula (II-a) or (II-b) with a coupling agent such as HBTU or EDCI in the presence of a base such as DIPEA and triethylamine , in a suitable reaction-inert solvent such as, for example, DCM or DMF, etc., at a suitable temperature, for example at room temperature; alternatively, intermediates of formula (II-a) or (II-b) can be reacted with a phosphonic anhydride, such as 1-propanephosphonic anhydride, in the presence of a suitable base, such as triethylamine, in a suitable reaction-inert solvent, such as, for example dry ACN. The carboxylic acids of formula (III) are either commercially available or can be prepared according to procedures known in the art.

Experimental procedure 2.

- 10 039788

Reaction scheme 2.

B: reaction with Ν,Ν-dimethylacetamide dimethyl acetal.

C, F: reaction with 1H-1,2,4-triazol-3-amine hydrochloride.

D: elimination of the protecting group.

E: reaction with 2,2-difluoroacetic acid ethyl ester.

The preparation of intermediates of formula (II-a), for example, where R ^A is methyl or CHF ₂ , referred to herein as intermediates of formula (II-a1) and (II-a2), respectively, can be provided from intermediates of formula ( IV-a), where PG is a suitable amino protecting group such as, for example, tert-butyloxycarbonyl (Boc).

The reaction with pure N,N-dimethylacetamide dimethylacetal can be carried out under temperature conditions such as, for example, heating at 100°C.

The reaction with 2,2-difluoroacetic acid ethyl ester can be carried out in the presence of a base such as KO ^t Bu in a reaction inert solvent such as toluene at a suitable temperature such as 0-5° C., then at r.t. .

The bicyclic core can be formed by reacting intermediates (V-a) or (V-b) with 1H-1,2,4-triazol-3-amine hydrochloride in a reaction inert solvent such as, for example, DMF, under temperature conditions such as such as heating at 80°C. In the case of (V-b)-(VI-b), the protecting group may be unstable and a subsequent step of introducing protective groups may be required.

Cleavage of the protecting group in intermediates (VI-a) or (VI-b) can be carried out according to procedures known in the art, for example, if the protecting group is Boc, the cleavage can be carried out under acidic conditions, such as, for example, HCl in MeOH at r.t. or TFA in DCM.

Experimental procedure 3.

- 11 039788

Reaction scheme 3.

G: reaction with carbon disulfide and methyl iodide.

H, L: reaction with 1H-1,2,4-triazol-3-amine hydrochloride.

I: oxidation.

J: reaction with NaCN.

K: reaction with N,N-dimethylformamide dimethyl acetal.

L: methylation.

D: elimination of the protecting group.

Intermediates of formula (II-a) where R ^A is cyano, herein referred to as (II-a3), or where R ^A is hydrogen and R ¹ is methyl, herein referred to as (II-a4), may be prepared according to a series of synthetic steps from intermediates of formula (IV-b) which are either commercially available or prepared according to procedures known in the art (eg R2=H or methyl, PG=Boc).

Thus, reaction with carbon disulfide and then with methyl iodide in the presence of a base such as NaH and a reaction inert solvent such as THF at 0°C provides (Vc) which can then be reacted with hydrochloride 1H-1 ,2,4-triazol-5-amine under the conditions described herein, thereby obtaining (VI-c, n=0), followed by oxidation with a suitable peroxyacid such as, for example, mCPBA , in the presence of a reaction-inert solvent such as, for example, DCM or CHCl ₃ , to give (VI-e, n=2). Substitution of the methyl sulfone group with a source of cyanide, such as, for example, NaCN in DMSO, provides (VII-c), which can be subjected to cleavage of the protective group under the conditions described herein.

The reaction of pure (IV-b) with N,N-dimethylformamide dimethyl acetal can be carried out under temperature conditions such as reflux. Reaction with 1H1,2,4-triazol-3-amine hydrochloride under the conditions described herein provides (VI-d) which can be methylated under conditions known in the art, such as, for example, by exposure its reaction with methyl iodide in the presence of a base such as NaH in a reaction inert solvent such as DMF. Subsequent cleavage of the protecting group under the conditions described herein provides intermediate (II-a4).

Experimental procedure 4.

Reaction scheme 4.

M: fluorination.

N: methylation and/or saponification.

X: recovery.

O: Suzuki reaction (alkylation) and saponification.

P: hydrogenation.

Q: saponification.

R: Weinreb amide formation.

S: conversion of amide to ketone (eg Grignard reaction).

The formation of intermediates (IV-a) or (IV-b) can be carried out by a series of functional group interconversions starting from intermediates (VIII), (IX) or (X), which are either commercially available or can be obtained, for example, in accordance with procedures such as those described herein.

Compounds of formula (VIII) wherein R ^2a is hydrogen or methyl and PG is Boc are either commercially available or prepared according to a number of known procedures such as those described herein. They may be fluorinated or alkylated according to procedures known in the art, for example by reaction with Nfluorobenzenesulfonimide in the presence of a base such as LDA in a reaction inert solvent such as THF, or by alkylation with an alkyl iodide after treatment with a base such as as LiHMDS; while optionally subsequent saponification under conditions known from the prior art provides (XI).

Compounds of formula (IX) are also known in the art and can be alkylated by Suzuki type procedures using conditions known to the person skilled in the art such as using boronic acid/ester in the presence of a catalyst such as Pd(PPh ₃ ) ₄ in a reaction inert solvent such as 1,4-dioxane under temperature conditions such as heating. Subsequent saponification and hydrogenation under conditions similar to those described herein provide intermediates of formula (XI).

The subsequent formation of the Weinreb amide and the conversion of the amide to the ketone according to the Grignard reaction as described herein provide the desired intermediates (IV-a) or (IV-b).

Experimental procedure 5.

Reaction scheme 5.

T: reaction with methyl cyanoformate.

U: recovery.

V: dehydration.

W: hydrogenation.

X: combination.

Y: decarboxylation and cleavage of the protecting group.

The formation of intermediates of formula (II-bc) where ---- is CH2 or CH ₂ CH ₂ can be obtained by a series of synthetic steps starting from commercially available starting materials of formula (XIV) such as N-Boc- nortropinone [185099-67-6] or 6-Boc-3-oxo-6azabicyclo[3.1.1]heptane [1246281-86-6]. Reaction with methyl cyanoformate in the presence of a base such as nBuLi and NH ⁱ Pr2 in a reaction inert solvent such as THF at a suitable temperature such as -78°C provides the ketoester (XV) which can then be reduced with using NaBH ₄ under conditions known in the art, for example in MeOH at about 0°C, and then dehydrate, for example, trifluoroacetic anhydride, in the presence of a base, such as triethylamine and DMAP, in a reaction-inert solvent, such as DCM, while maintaining temperatures below 60°C. Hydrogenation under conditions known in the art, such as, for example, in the presence of palladium on carbon in MeOH as a catalyst, provides an intermediate compound (XVIII), which can then be reacted with intermediates of formula (XIX), which are either commercially available or prepared according to procedures known in the art in the presence of a base such as, for example, LDA in a reaction inert solvent such as THF at -78 to -60°C. Reaction with concentrated HCl under temperature conditions, such as, for example, heating at 150°C, provides intermediate compound (II-bc) with simultaneous removal of the protective group if it is acid-labile, such as, for example, Boc. An alternative route for preparing an intermediate of formula (II-bc) wherein R ^A is CHF2 and --- is CH ₂ CH ₂ from a commercially available starting material is described herein in the examples.

Pharmacology

The compounds of the present invention inhibit the activity of the PDE2 enzyme, in particular PDE2A, and consequently increase cAMP or cGMP levels in cells that express PDE2. Accordingly, inhibition of PDE2 enzyme activity may be useful in the treatment of diseases caused by insufficient amounts of cAMP or cGMP in cells. PDE2 inhibitors may also be useful in cases where increasing cAMP or cGMP above normal levels provides a therapeutic effect. PDE2 inhibitors can be used to treat neurological and psychiatric disorders.

Therefore, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof according to the present invention for use as a drug, as well as to the use of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof according to the present invention, or a pharmaceutical composition according to the present invention for the manufacture of a medicinal product. The present invention also relates to a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof according to the present invention, or to a pharmaceutical composition according to the present invention for use in the treatment or prevention, in particular in the treatment, of a condition in a mammal, including a person whose treatment or prevention depends on, or is facilitated by, inhibition of the phosphodiesterase 2 enzyme. The present invention also relates to the use

- 14 039788 a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof according to the present invention, or a pharmaceutical composition according to the present invention for the manufacture of a medicament for the treatment or prevention, in particular the treatment, of a condition in a mammal, including a human, treatment or the prevention of which is dependent on, or facilitated by, inhibition of the phosphodiesterase 2 enzyme.

The present invention also relates to a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof according to the present invention, or a pharmaceutical composition according to the present invention for use in the treatment, prevention, amelioration, control or reduction of the risk of various neurological and mental disorders associated with dysfunction of phosphodiesterase 2 in a mammal, including humans, the treatment or prevention of which depends on inhibition of phosphodiesterase 2 or is facilitated by it.

In addition, the present invention relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof according to the present invention, or a pharmaceutical composition according to the present invention, for the manufacture of a medicament for the treatment, prevention, amelioration, control or reduction of the risk of developing various neurological and psychiatric disorders associated with dysfunction of phosphodiesterase 2 in a mammal, including humans, the treatment or prevention of which depends on inhibition of phosphodiesterase 2 or is facilitated by it.

When it is said that the present invention relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, or a composition according to the present invention for the manufacture of a medicinal product, for example, for the treatment of a subject, for example a mammal, it is understood that in certain areas such use should be understood as a method of, for example, treating a subject, comprising administering to a subject in need of such, for example, treatment, an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, or a composition according to the present invention.

In particular, indications that can be treated with PDE2 inhibitors alone or in combination with other drugs include, without limitation, diseases believed to be mediated in part by the basal ganglia, prefrontal cortex, and hippocampus.

These indications include neurological and psychiatric disorders selected from psychotic disorders and conditions; anxiety disorders; movement disorders; drug addiction; affective disorders; neurodegenerative disorders; disorders or conditions including attention deficit disorder and/or cognitive impairment as a symptom; disorders associated with memorization and memory consolidation; stroke and autistic disorder or autism.

In particular, psychotic disorders and conditions associated with impaired PDE2 function include one or more of the following conditions or diseases: schizophrenia, eg paranoid, disorganized, catatonic, undifferentiated or residual type; schizophreniform disorder; a schizoaffective disorder such as, for example, the delusional or depressive type; delusional disorder; psychotic disorder caused by the use of certain substances, such as psychosis caused by the use of alcohol, amphetamine, marijuana, cocaine, hallucinogenic substances, volatile substances of narcotic action, opioids or phencyclidine; personality disorders of the paranoid type; and personality disorder of the schizoid type.

In particular, anxiety disorders include panic disorder; agoraphobia; specific phobia, social phobia; obsessive-compulsive disorder; post-traumatic stress disorder, acute stress disorder, and generalized anxiety disorder.

In particular, movement disorders include Huntington's disease and dyskinesia; Parkinson's disease; restless leg syndrome and essential tremor. In addition, Tourette's syndrome and other tic disorders may be included.

In particular, the central nervous system disorder is a substance use disorder selected from the group of alcohol abuse; alcohol addiction; alcohol withdrawal syndrome; alcohol withdrawal syndrome with delirium; alcohol-induced psychotic disorder; amphetamine addiction; amphetamine withdrawal syndrome; cocaine addiction; cocaine withdrawal syndrome; nicotine addiction; nicotine withdrawal syndrome; opioid addiction and opioid withdrawal syndrome.

In particular, affective disorders and affective episodes include depression, mania, and bipolar disorders. Preferably, the affective disorder is selected from the group of bipolar disorders (types I and II); cyclothymic disorder; depression; dysthymic disorder; major depressive disorder; treatment-resistant depression; and mood disorder caused by the use of certain substances.

In particular, neurodegenerative disorders include Parkinson's disease; Hunting disease - 15 039788 tone; dementia, such as, for example, Alzheimer's disease; multi-infarct dementia; AIDS-associated dementia or frontotemporal dementia. The neurodegenerative disorder or condition involves dysfunction in the responses of the striatal medium spiny neurons.

In particular, disorders or conditions that include attention deficit disorder and/or cognitive impairment as a symptom include dementia such as Alzheimer's disease; multi-infarct dementia; dementia due to Lewy body disease; alcohol dementia or persistent dementia caused by the use of certain substances; dementia associated with intracranial tumors or traumatic brain injury; dementia associated with Huntington's disease; dementia associated with Parkinson's disease; AIDS-associated dementia; dementia due to Pick's disease; dementia due to Creutzfeldt-Jakob disease; other diseases including delirium; amnestic disorder; post-traumatic stress disorder; stroke; progressive supranuclear palsy; mental retardation; impaired ability to learn; attention deficit/hyperactivity disorder (ADHD); mild cognitive impairment; Asperger's syndrome; age-related cognitive impairment; and cognitive impairment associated with perception, attention, learning, or memory.

In particular, memory disorders and memory consolidation include memory disorders such as, for example, age-related memory loss, amnesia.

Preferably, the psychotic disorder is selected from the group of schizophrenia, delusional disorder, schizoaffective disorder, schizophreniform disorder, and substance use disorder.

Preferably, the central nervous system disorder is a personality disorder selected from the group of obsessive-compulsive personality disorder and schizoid, schizotypal disorder.

Preferably, the central nervous system disorder is an affective disorder selected from the group of bipolar disorders (type I and II), cyclothymic disorder, depression, dysthymic disorder, major depressive disorder; treatment-resistant depression; and mood disorder caused by the use of certain substances.

Preferably, the central nervous system disorder is attention deficit/hyperactivity disorder.

Preferably, the central nervous system disorder is a cognitive disorder selected from the group of delirium, persistent delirium caused by the use of certain substances, dementia, dementia due to HIV disease, dementia due to Huntington's disease, dementia due to Parkinson's disease, dementia of the Alzheimer's type, persistent dementia caused by the use of certain substances, and mild cognitive impairment.

Preferably, the disorders to be treated with the compounds of formula (I) or a pharmaceutically acceptable salt or solvate thereof of the present invention are selected from schizophrenia; obsessive-compulsive disorder; generalized anxiety disorder; Huntington's disease; dyskinesia; Parkinson's disease; depression; bipolar disorders; dementia such as Alzheimer's disease; attention deficit/hyperactivity disorder; drug addiction; stroke and autism.

Preferably, the disorders to be treated with the compounds of formula (I) or a pharmaceutically acceptable salt or solvate thereof of the present invention are schizophrenia, including its positive and negative symptoms, as well as cognitive impairments such as attention or memory impairment.

Among the disorders mentioned above, of particular importance is the treatment of anxiety, obsessive-compulsive disorder, post-traumatic stress disorder; Generalized Anxiety Disorder, Schizophrenia, Depression, Attention Deficit/Hyperactivity Disorder, Alzheimer's Disease, Huntington's Disease Dementia, Parkinson's Disease Dementia, Alzheimer's Type Dementia, Persistent Substance Use Dementia, and Mild Cognitive Impairment.

Among the disorders mentioned above, the treatment of anxiety, obsessive-compulsive disorder, schizophrenia, depression, attention deficit/hyperactivity disorder and Alzheimer's disease is of particular importance.

Other central nervous system disorders include schizophrenia-associated anxiety disorder and comorbid depression and anxiety, in particular major depressive disorder with comorbid generalized anxiety disorder, social anxiety disorder, or panic disorder; it should be understood that comorbid depression and anxiety may also be referred to by the terms depression accompanied by anxiety, mixed anxiety and depression, mixed anxiety and depressive disorder, or major depressive disorder with anxiety symptoms, which are used herein without distinction.

- 16 039788

The American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) fourth edition now provides a diagnostic method for identifying the disorders described herein. One of skill in the art will appreciate that alternative nomenclature systems, nosological approaches, and classification systems exist for the neurological and psychiatric disorders described herein, and that they evolve with advances in the medical field and scientific progress. For example, in the American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition. Arlington, VA, American Psychiatric Association, 2013 (DSM-5™) uses terms such as depressive disorders, specifically major depressive disorder, persistent depressive disorder (dysthymia), substance/drug use depressive disorder; neurocognitive disorders (NCD) (both severe and mild), in particular neurocognitive disorders due to Alzheimer's disease, vascular NCD (such as vascular NCD manifesting as multiple infarcts), NCD due to HIV infection, NCD due to trauma brain injury (TBI), NCD due to Parkinson's disease, NCD due to Huntington's disease, frontotemporal NCD, NCD due to prion disease, and NCD due to the use of certain substances/drugs; neurodevelopmental disorders, in particular intellectual disability, specific learning disability, motor neurodevelopmental disorder, communication disorder, and attention deficit/hyperactivity disorder (ADHD); substance use disorders and addiction disorders, in particular alcohol use disorder, amphetamine use disorder, marijuana use disorder, cocaine use disorder, other hallucinogenic substance use disorder , tobacco use disorder, opioid use disorder, and phencyclidine use disorder; schizophrenia spectrum disorders and other psychotic disorders, in particular schizophrenia, schizophreniform disorder, schizoaffective disorder, delusional disorder, brief psychotic disorder, substance/drug induced psychotic disorder; and cyclothymic disorder (which, according to the DSM-5™, falls under the category of bipolar and related disorders). Such terms may be used by one of skill in the art as an alternative nomenclature for some of the diseases or conditions referred to herein. Additional neurodevelopmental disorder includes Autism Spectrum Disorder (ASD), which according to the DSM-5™ covers disorders formerly known as early childhood autism, childhood autism, Kanner's autism, high-functioning autism, atypical autism, pervasive developmental disorder without further specification , disintegrative disorder of childhood and Asperger's syndrome.

Therefore, the present invention also relates to a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof according to the present invention for use in the treatment of any of the diseases mentioned herein.

The present invention also relates to a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof according to the present invention for use in the treatment of any of the diseases mentioned herein.

The present invention also relates to a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof according to the present invention for the treatment or prevention, in particular treatment, of any of the diseases mentioned herein.

The present invention also relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof according to the present invention for the manufacture of a medicament for the treatment or prevention of any of the disease states mentioned herein above.

The present invention also relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof according to the present invention for the manufacture of a medicament for the treatment of any of the disease states mentioned herein above.

A compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof of the present invention may be administered to mammals, preferably humans, for the treatment or prevention of any of the diseases mentioned herein above.

In view of the utility of the compounds of formula (I) and their pharmaceutically acceptable salts and solvates of the present invention, there is provided a method for treating a disorder or disease as defined herein, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I), or pharmaceutically acceptable salt or solvate, or pharmaceutical compositions described herein.

These methods involve the introduction, ie. systemic or topical administration, preferably oral administration, of a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof according to the present invention to warm-blooded animals, including humans.

Therefore, the present invention also relates to a method for preventing and/or treating any of the diseases mentioned herein, comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof according to the present invention to a patient in need thereof.

The PDE2 inhibitor described herein may be used alone, in combination, or in combination with other pharmaceutical agents, such as other agents used in the treatment of psychoses such as schizophrenia and bipolar disorder, obsessive-compulsive disorder, Parkinson's disease, cognitive impairment and/or memory loss, eg, α-7 nicotinic acetylcholine receptor agonists, PDE4 inhibitors (rolipram, GEBR-7b, GSK356278, GSK256066, apremilast, MK-0952, roflumilast, AN2898, AN2728, Ariflo (cilomilast), dotraverine, ronomilast (elbimilast), revamilast , tetomilast, E6005, GDP-1116, HT0712, MK-0873), PDE5 inhibitors (sildenafil, vardenafil, tadalafil, udenafil, avanafil, mirodenafil, lodenafil, dazantafil, PF-00489791), PDE9 (PF-04447943), other PDE2 inhibitors (Bay 60-7550, PF-999, ND-7001), PDE10 inhibitors (PF-02545920, AMG579), PDE2 and 10 inhibitors, calcium channel blockers, m1 and m2 muscarinic acetylcholine receptor modulators, ade modulators nosine receptor modulators, ampakines, NMDA-R modulators, mGluR modulators, dopamine receptor modulators, serotonin receptor modulators, cannabinoid receptor modulators, HDAC inhibitors (vorinostat (SAHA), panobinostat, quisinostat, valproic acid) and cholinesterase inhibitors (eg donepezil, rivastigmine and galantamine). In such combinations, a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, of the present invention may be used in combination with one or more other drugs in the treatment, prevention, control, amelioration, or risk reduction of diseases or conditions, in for which the compound of formula (I) or other drugs may be useful, and the combination of drugs taken together is safer or more effective than each of the drugs alone.

One skilled in the art will appreciate that a therapeutically effective amount of a PDE2 inhibitor of the present invention is an amount sufficient to inhibit the PDE2 enzyme, and that this amount varies, among other things, depending on the type of disease, the concentration of the compound in the therapeutic formulation, and the condition of the patient. Typically, the amount of PDE2 inhibitor to be administered as a therapeutic agent for diseases in which PDE2 enzyme inhibition is beneficial, such as the disorders described herein, will be determined on a case-by-case basis by the attending physician.

A suitable dose is usually one that results in a PDE2 inhibitor concentration in the treated area in the range of 0.5 nM to 200 μM, and more typically 5 nM to 50 μM. To achieve these therapeutic concentrations, a patient in need of treatment will likely be administered from 0.001 to 15 mg/kg of body weight, in particular from 0.01 to 2.50 mg/kg of body weight, in particular from 0.01 to 1, 5 mg/kg body weight, in particular 0.1 to 0.50 mg/kg body weight. The amount of a compound of the present invention, also referred to herein as an active ingredient, required to achieve a therapeutic effect will, of course, vary on a case-by-case basis, vary with the particular compound, route of administration, age and condition of the patient being treated, and specific disorder or disease to be treated. The method of treatment may also include administering the active ingredient according to a regimen of one to four administrations per day. In such treatments, the compound of the present invention is preferably formulated prior to administration. As described herein below, suitable pharmaceutical formulations are prepared by known procedures using well known and commonly available ingredients.

Pharmaceutical compositions

The present invention also provides compositions for the prevention or treatment of diseases in which PDE2 inhibition is beneficial, such as neurological and psychiatric disorders. Said compositions contain a therapeutically effective amount of a compound of formula (I) and a pharmaceutically acceptable carrier or diluent.

While the active ingredient may be administered alone, it is preferred if it is presented as a pharmaceutical composition. Therefore, the present invention further provides a pharmaceutical composition comprising a compound of the present invention together with a pharmaceutically acceptable carrier or diluent. The carrier or diluent must be acceptable in terms of compatibility with the other ingredients of the composition and must not be harmful to the patients receiving it.

The pharmaceutical compositions of the present invention may be prepared by any of the methods well known in the pharmaceutical art. A therapeutically effective amount of a particular compound, in base or addition salt form, as the active ingredient is combined in a homogeneous mixture with a pharmaceutically acceptable carrier which may take a number of forms depending on the form of preparation desired for administration. Desirably, these pharmaceutical compositions are in unit dosage form, preferably suitable for systemic administration such as oral, transdermal or parenteral administration; or for topical administration, such as by inhalation, nasal spray, eye drops, or by cream, gel, shampoo, and the like. For example, when preparing compositions in the form of a dosage form for oral administration, any conventional pharmaceutical vehicles can be used, such as, for example, water, glycols, oils, alcohols, etc., in the case of liquid preparations for oral administration, such as suspensions, syrups , tinctures and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrants and the like, in the case of powders, pills, capsules and tablets. Because of their ease of administration, tablets and capsules are the most preferred oral dosage unit forms, in which case solid pharmaceutical carriers are undoubtedly used. In the case of compositions for parenteral administration, the carrier will typically contain at least a substantial amount of sterile water, although other ingredients may be included, for example, to aid solubility. For example, injectable solutions can be prepared in which the carrier contains saline solution, glucose solution, or a mixture of saline and glucose solution. Injectable suspensions can also be prepared, in which case appropriate liquid carriers, suspending agents, and the like can be used. In compositions suitable for transdermal administration, the carrier optionally contains a penetration enhancer and/or a suitable wetting agent, optionally in combination with suitable additives of any nature in minimal proportions, without the additives causing any significant adverse effects on the skin. These additives may facilitate the administration through the skin and/or may be useful in preparing the desired compositions. These compositions can be administered in various ways, for example, via a transdermal patch, by topical application, or as an ointment.

It is particularly preferred to formulate the above pharmaceutical compositions in unit dosage form for ease of administration and dosage uniformity. Unit dosage forms, as used herein and in the claims, refer to physically discrete units suitable as dosage units, each unit containing a predetermined amount of the active ingredient calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including divisible or coated tablets), capsules, pills, pouches of powdered product, strips, injection solutions or suspensions, heaped teaspoons, heaped tablespoons, and the like. ., as well as their individual multiples.

Depending on the route of administration, the pharmaceutical composition will contain from 0.05% to 99% by weight, preferably from 0.1% to 70% by weight, more preferably from 0.1% to 50% by weight of the active ingredient and from 1% up to 99.95% by weight, preferably from 30% to 99.9% by weight, more preferably from 50% to 99.9% by weight of a pharmaceutically acceptable carrier, with all percentages based on the total weight of the composition.

The compound of the present invention can be used for systemic administration such as oral, transdermal or parenteral administration; or for topical administration, such as by inhalation, nasal spray, eye drops, or by cream, gel, shampoo or the like. The compound is preferably administered orally.

The precise dosage and frequency of administration will depend upon the compound, the particular condition being treated, the severity of the condition being treated, the age, weight, sex, severity of the disorder, and the general physical condition of the particular patient, as well as other medical treatments the individual may be receiving, which well known to those skilled in the art. Moreover, it is clear that said effective daily amount may be reduced or increased depending on the response of the subject being treated and/or depending on the judgment of the physician prescribing the compound of the present invention.

The amount of a compound of formula (I) that can be combined with a carrier material to form a single dosage form will vary depending on the disease being treated, the species of mammal, and the particular route of administration. However, as a general guideline, suitable unit doses of a compound of the present invention may, for example, preferably contain from 0.1 mg to about 1000 mg of the active compound. A preferred unit dose is from 1 mg to about 500 mg. A more preferred unit dose is from 1 mg to about 300 mg. An even more preferred unit dose is from 1 mg to about 100 mg. Such unit doses can be administered more than once a day, for example 2, 3, 4, 5 or 6 times a day, but preferably 1 or 2 times a day, so that the total dosage for a 70 kg adult is in the range 0.001 to approx.

- 19,039,788 mg per kg of subject weight per administration. The preferred dose is from 0.01 to about 1.5 mg per kg of subject weight per administration, and such therapy may be continued for several weeks or months, and in some cases for several years. However, it should be understood that the specific dosage level for any particular patient will depend on a number of factors, including the potency of the particular compound used; age, body weight, general health, sex and diet of the individual being treated; time and route of administration; withdrawal speed; other medicines that have been introduced previously; and the severity of the particular disease being treated, which is well understood by those skilled in the art.

A typical dosage may be one tablet containing 1 mg to about 100 mg or 1 mg to about 300 mg taken once a day or several times a day, or one slow release capsule or tablet taken once a day and characterized by a proportionately higher content of the active ingredient. The slow release effect can be achieved with capsule materials that dissolve at various pH values, with slow release capsules under osmotic pressure, or with any other known controlled release means.

In some cases, it may be necessary to use dosages outside these ranges, which will be obvious to specialists in this field. In addition, it should be noted that the clinician or physician will know how and when to start, interrupt, adjust or terminate therapy according to the individual patient's response.

With respect to the compositions, methods, and kits above, one of ordinary skill in the art will appreciate that the preferred compound for use in each is the one identified herein.

experimental part

As used herein, ACN means acetonitrile, AcOH or TFA means acetic acid, Boc means tert-butyloxycarbonyl, Boc2O means di-tert-butyl dicarbonate, d means day(s), DMAP means 4-dimethylaminopyridine, DSC means Differential Scanning Calorimetry, LCMS means liquid chromatography/mass spectrometry, HPLC stands for High Performance Liquid Chromatography, RP HPLC stands for Reversed Phase High Performance Liquid Chromatography, aq. means aqueous, DCM means dichloromethane, DIPE means diisopropyl ether, DIPEA means diisopropylethylamine, DMF means N,N-dimethylformamide, DMSO means dimethyl sulfoxide, EDCI means 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EtOH means ethanol, Et2O means diethyl ether, EtOAc means ethyl acetate, Et ₃ N or TEA means triethylamine, HATU means 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate, HBTU means O-(benzotriazol-1-yl)-N,N,NN,'-tetramethyluronium hexafluorophosphate, LiHMDS means lithium bis(trimethylsilyl)amide, THF means tetrahydrofuran, min means minutes, h means hours, mCPBA means 3-chloroperbenzoic acid , MeOH means methanol, MTBE means methyl tert-butyl ether, Pd/C means palladium on carbon, Pd(PPh ₃ ) ₄ means tetrakis(triphenylphosphine)palladium(0), iPrOH means 2-propanol, RM or reakts. see means reaction mixture, K.T. or K.T. means room temperature, OL means organic layer, R _t means retention time (in minutes), no. means quantitative, sat. means saturated, SFC means supercritical fluid chromatography, rast. means solution, so pl. means melting point, qs means enough.

Thin layer chromatography (TLC) was carried out on plates with a layer of silica gel 60 F254 (Merck) using chemically pure solvents. Open column chromatography was performed on silica gel with a particle size of 230-400 mesh and a pore size of 60 A (Merck) according to standard procedures.

Automated flash column chromatography was performed using Merck's ready-to-plug 15-40 µm irregular particle size silica gel cartridges (disposable normal phase flash chromatography columns) on an Armen Instrument SPOT or LAFLASH system.

If the stereocenter is designated RS, this means that a racemic mixture with the specified center was obtained, unless otherwise indicated. The stereochemical configuration of the centers for a number of compounds could be designated as R or S if the mixture(s) was(were) separated(s); for some compounds, the stereochemical configuration at the indicated centers was designated as *R or *S if the absolute stereochemical configuration was not determined, although the compound itself was isolated as a single stereoisomer and is enantiomerically/diastereomerically pure.

The absolute stereochemical configuration for some compounds was determined using vibrational circular dichroism (VCD). They were measured on a Bruker Equinox 55 equipped with a PMA 37 in a liquid cell with a KBr window using CD2C12 as solvent (PEM: 1350 cm ^-1 , LIA: 1 mV, resolution: 4 cm ^-1 ). A description of the use of VCD to determine the absolute configuration can be found in Dyatkin A.V. et. al., Chirality, 14:215-219 (2002). Calculations ab initio: a thorough search in relation to

- 20 039788 The conformation was performed at the level of molecular mechanics using a Macromodel to perform sampling in mixed torsion/low frequency mode using an OPLS-2005 force field. Localized minima were optimized using Jaguar at the B3LYP/6-31G** level in a Poisson-Boltzmann continuum solvation model to simulate the dichloromethane solvent. All conformations within the 10 kJ/mol range were used to simulate the VCD spectrum and the IR spectrum. Dipole and rotational forces were calculated at the same B3LYP/6-31G** level using Jaguar. The calculated VCD spectra obtained after frequency scaling by a factor of 0.97, Lorentz transform to band shape and summation of the contribution of each conformer, taking into account the Boltzmann ensemble, were visually compared with the experimental spectra to determine the exact stereochemistry.

One of skill in the art will recognize that compounds synthesized using these protocols may exist as a solvate, eg, hydrate, and/or may contain residual solvent or minor impurities. Compounds isolated in the form of a salt can be characterized by an integer stoichiometric index, i.e. be mono or disalts, or an intermediate stoichiometric index.

The following examples are intended to illustrate, but not to limit the scope of the present invention. Unless otherwise indicated, all starting materials were obtained from commercial suppliers and used without further purification.

A. Synthesis of intermediates.

Intermediate 1.

Procedure a. 4-Methyl-3-pyridinecarboxylic acid hydrochloride (1:1) (40 g, 230.4 mmol) was added to a refluxed mixture of sulfuric acid (20 ml) and MeOH (400 ml). The mixture was heated at reflux overnight, then it was evaporated and the resulting slurry was added to a cold solution of NaHCO ₃ (64 g) in water (360 ml). The product was extracted with DCM and OL dried over MgSO ₄ , filtered and evaporated to give Intermediate 1 (28.70 g, 83%).

procedure b. A metal reactor was charged with 3-bromo-4-methylpyridine (200 g, 0.116 mol) and a DMF/MeOH mixture (1 L/1 L). Et ₃ N (400 g, 0.395 mol), palladium(II) acetate (8 g, 0.036 mol) and 1,1'-bis(diphenylphosphino)ferrocene (16 g, 0.029 mol) were added to this. The reactor was closed and gaseous CO (3 MPa) was supplied, and the reaction mixture was stirred and heated overnight at 140°C. reaction see cooled, filtered and concentrated in vacuo. The residue was purified by silica gel flash column chromatography (elution gradient: EtOAc/petroleum ether, 1/1 to 1/0). The product fractions were collected and the solvent was evaporated to give the desired intermediate 1 (90 g, 51%).

Intermediate 2.

Procedure a. A hydrogenation flask was charged with AcOH (500 ml) and then PtO ₂ (15.02 g, 66.2 mmol) was added. Intermediate 1 (50 g, 330.8 mmol) was added and the mixture was hydrogenated at 50° C. for 7 days. reaction see filtered through dicalite® and the filtrate was evaporated to give intermediate 2 (52 g) which was used in the next step without further purification.

Procedure b. Platinum oxide (5 g, 0.022 mol) was added to a solution of intermediate 1 (90 g, 0.595 mol) and AcOH (1 L). reaction see stirred and hydrogenated for 5 days at 50°C at a pressure of 3.5 kPa. chilled reaction. see concentrated in vacuo to give intermediate 2 as the acetic acid salt (140 g, 97%, 90% purity, determined by 1H NMR).

- 21 039788

Intermediate 3.

O

About I

Procedure a. To a solution of intermediate 2 (52 g, 330.8 mmol) in DCM (869 ml) was added DIPEA (85.5 g, 661.5 mmol) and DMAP (4.04 g, 33.08 mmol). Di-tert-butyl dicarbonate (72.19 g, 330.8 mmol) was then added in small portions to this solution and the reaction mixture was stirred at rt. within 1 hour Reaction. see washed with water and brine and the organic layer was dried over MgSO ₄ , filtered and evaporated. The product was purified by column chromatography (silica gel, eluent: DCM, 1% MeOH in DCM, 2%, 4%). The desired fractions were evaporated to give intermediate 3 (64.1 g, 75%).

procedure b. To a stirred and cooled (0°C) solution of intermediate 2 (140 g, 0.595 mol) in DCM (1.5 L) was added successively di-tert-butyl dicarbonate (130 g, 0.596 mol), Et ₃ N (225 g, 1.74 mol) and DMAP (10 g, 0.082 mol) and stirring was continued at rt. within 2 hours the Reaction mixture was poured into H ₂ About (500 ml) and was extracted with DCM (2x100 ml). The organic layers were separated, dried (Na ₂ SO ₄ ) and the solvent was evaporated to give the crude intermediate 3 (150 g, 90%, 90% purity, determined by 1H NMR), which was used further as such.

Intermediate 4.

⁰ I

BUT

Procedure a. Intermediate 3 (64.1 g, 249.1 mmol) was stirred in MeOH (500 mL) at rt. NaOH (2 M, 747.3 ml) was added and the mixture was stirred for 2 hours at RT. reaction see acidified with 1 u. HCl and product were extracted with Et ₂ O. OL was washed with brine and dried over MgSO ₄ , filtered and evaporated to give Intermediate 4 (59.70 g) as a white solid.

procedure b. To a stirred solution of intermediate 3 (150 g, 90% purity, 0.524 mol) in MeOH (0.9 L) was added 2 M NaOH (1.8 mol). After 14 hours, the reaction see extracted at rt. using MTBE (2x0.8 l). The aqueous layer was acidified with 10% citric acid and then extracted with EtOAc (4x1 L). The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo to give crude intermediate 4 (142 g, 90% purity, determined by 1 H NMR, 100%), which was used as such in the next step.

Intermediate 5.

Procedure a. To a solution of intermediate 4 (59.7 g, 0.25 mol) in THF (800 ml) was added di-1H-imidazol-1-ylmethanone (54 g, 0.33 mol) and the mixture was stirred at rt. for 1 hour. In another flask, trimethylamine (35.75 g, 0.35 mol) was added to a suspension of N-methoxymethanamine hydrochloride (1:1) (32.93 g, 0.34 mol) in ACN (500 ml). Both mixtures were combined and stirred at 50° C. while monitoring. The intermediate product was crystallized from the reaction. see, and it did not react with N-methoxymethanamine to form the desired product. DCM was added until the intermediate was dissolved. The reaction mixture was allowed to stir for 1 week at 80°C. The solvents were evaporated. The residue was dissolved in DCM and washed with water, 20% AcOH solution and finally with saturated NaHCO ₃ solution. OL was dried over MgSO ₄ , filtered and evaporated. The product was purified by column chromatography (silica gel, eluent: 2% MeOH in

-22039788

DCM, 4%). Purified fractions were evaporated to give intermediate 5 (70 g, quantitatively).

Procedure b. To a stirred and ice-cold solution of intermediate 4 (140 g, 0.518 mol) in DCM (2 L) was added N-O-dimethylhydroxylamine (113 g, 1.16 mol) and Et ₃ N (113 g, 1.79 mol). HATU (235 g, 0.618 mol) was then added and stirring was continued for 14 h. The solvent was evaporated and NaHCO ₃ solution (0.5 L) was added and then extracted with DCM (3x1 L). The combined organic layers were separated, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel flash chromatography eluting with 1 to 10% EtOAc in petroleum ether to give Intermediate 5 (152 g, 100%).

Intermediate 6.

Procedure a. Loaded intermediate compound 5 (70 g, 244.4 mmol) in THF (250 ml) in a flask under N 2 atmosphere and cooled to -15°C. Methylmagnesium bromide (1.4 M in toluene/THF, 75/25, 206 ml) was added dropwise at a temperature not exceeding 0°C. After adding react. see stirred at room temperature for 1 hour. Then the reaction. see poured onto ice with 20 ml AcOH. The product was extracted with Et2O and OL was washed with 5% NaHCO3 solution. OL was dried over MgSO4, filtered and evaporated to give intermediate 6 (53.35 g, 90%).

procedure b. To a stirred and cooled solution (0°C) of intermediate 5 (150 g, 0.524 mol) in THF (2 L) was added dropwise a 3 M solution of methylmagnesium bromide in THF (0.75 L, 2.25 mol) and stirring was continued at k.t. for 2 hours. The reaction mixture was poured into aqueous NH4Cl and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with 1-5% EtOAc in petroleum ether to give Intermediate 6 (120 g, 95%).

Intermediate 7.

Intermediate 6 (53.35 g, 0.22 mol) was stirred in toluene (1500 ml) at 0° C. under N2. Potassium tert-butoxide (34.14 g) was added at 0-5°C and 2,2-difluoroacetic acid ethyl ester (33.01 g, 0.27 mol) was added dropwise at 0-5°C. reaction see stirred at rt. for 2 h, then washed with 10% H ₂ SO ₄ in water and OL dried over MgSO ₄ , filtered and evaporated to give intermediate 7 (70.50 g, quantitatively).

Intermediate 8.

Intermediate 7 (70.5 g, 220.8 mmol), 1H-1,2,4-triazol-5amine hydrochloride (1:1) (53.22 g, 441.52 mmol) and DMF (1500 ml) were stirred at 80°C for 24 h. Added Et ₃ N (20 g) and di

- 23 039788 tert-butyl dicarbonate (20 g). The mixture was stirred for 30 min, evaporated and then dissolved in EtOAc, washed with water and brine. OL was dried over MgSO ₄ , filtered and evaporated. Four isomers were observed. The first fraction was crystallized from Et ₂ O. The crystals were filtered off and dried to give intermediate 8 (24.60 g, 30%). A second fraction of the compound was obtained from the mother liquor. The crystals were filtered off and dried to give intermediate 8 (2.53 g, 3%).

Note: RS means that the intermediate is a racemic mixture of two enantiomers with a relative trans configuration.

Intermediates 9, 9a and 9b.

To a solution of intermediate 8 (24.6 g, 67 mmol) in MeOH (350 ml) was added HCl-iPrOH (350 ml) and react. cm. stirred for 2 h at room temperature. reaction see evaporated and the product was crystallized from EtOH. The crystals were filtered off and dried to give 20.33 g of a crude substance, to which water, Na ₂ CO ₃ and DCM were added. OL was dried over MgSO ₄ , filtered and evaporated to give 12.80 g of intermediate 9. This free base was separated into enantiomers 9a and 9b by purification with preparative SFC (stationary phase: Chiralpak Diacel AD 30x250 mm; mobile phase: CO ₂ . ((MeOH - iPrOH 50/50) with 0.4% iPrNH ₂ ) to give intermediate 9a (5 g, 19%, R _t = 7.57 min) and intermediate 9b (5.13 g, 19%, R _t =9.36 min).

Intermediates 9a and 9b were isolated as free bases or alternatively they were dissolved in MeOH followed by addition of HCl/i-PrOH and the mixture was evaporated. The hydrochloride salts (in each case .HCl) were crystallized from ACN, filtered off and dried.

Intermediate 10.

A stirred mixture of I-6 (7.3 g, 0.03 mol) in N,N-dimethylacetamide dimethyl acetal (20 ml, 0.91 g/ml, 0.14 mol) was heated at 100°C for 4 h. . see concentrated in vacuo, co-evaporated with toluene (2 x 20 ml) to give I-7 as a brown residue (9.4 g, 100.1% yield) which was used as such in the next step.

Intermediate 11 (I-11).

To a mixture of I-10 (9.4 g, 0.03 mol) in AcOH (50 ml, 1.05 g/ml, 1.75 mol) was added a mixture of 3-amino-1,2,4-triazole (2.68 g , 0.03 mol) in HOAc (50 ml, 1.05 g/ml, 1.75 mol) and heated the reaction. see Drysyn® metal heating block at 130°C for 15 min. see cooled, concentrated in vacuo, diluted with DCM (0.2 l) and treated with 1 NaOH to pH~8. The layers were separated and the aqueous layer was extracted with DCM (2x50 ml). The combined organic layers were dried (MgSO ₄ ), filtered and concentrated in vacuo to give a dark brown oil which was purified by silica gel chromatography using a Redisep® 120 g flash chromatography column, eluting with a gradient of 0 to 3% 7N . NH ₃ /MeOH in DCM to give intermediate 11 as a beige oil as a mixture of cis:trans isomers = ~1:4 (2.15 g, 21.42% yield).

- 24 039788

Intermediate 12.

A stirred mixture of I-11 (2.15 g, 0.0065 mol) in MeOH (50 ml, 0.79 g/ml, 1.23 mol) was treated with HCl (6 M in iPrOH) (50 ml, 6 M, 0.3 mol) and after 16 h at room temperature reaction see concentrated in vacuo to give an off-white solid. This was triturated with a mixture of Et2O (200 ml) and ACN (30 ml) for 16 h. The solid was collected by filtration and dried to give intermediate 12 as an off-white solid as a mixture of cis/trans isomers (18%/82 %) (1.7 g, 97.87% yield).

Intermediate 12a and Intermediate 12b.

2HCI

1-12a

1-12b

A stirred mixture of I-11 (23 g, 0.0694 mol) in MeOH (165 ml) was treated with HCl (6 M in iPrOH) (165 ml, 6 M, 0.986 mol) and after 16 hours at rt. reaction see concentrated in vacuo to give an off-white solid. This was diluted with water and DCM and treated with Na ₂ CO ₃ . OL was dried over MgSO ₄ , filtered and concentrated in vacuo to give a residue which was purified using SCF (stationary phase: Chiralpak® Diacel AD 20x250 mm, mobile phase: CO ₂ , MeOH-iPrOH (50-50)+0.4% iPrNH2) to give intermediates 12a and 12b i. They were dissolved in MeOH (100 ml) and treated with HCl (6 M in iPrOH) (100 ml) at 0°C for 2 h. The volatiles were evaporated under reduced pressure and the resulting residues were stirred at 0°C in Et ₂ O to give Intermediate 12a (9.25 g, 43%, R _t =3.54 min, [a] ²⁰ D=-17.47° (c 0.54, DMF)) and Intermediate 12b (8.8 d, 42%, Rt=3.24 min, [a] ²⁰ D=+16.5°(c 0.52, DMF)).

Intermediate 13.

A solution of intermediate 3 (20 g, 77.721 mmol) in THF ( 50 ml) at 0°C. The solution was stirred for 30 min and then transferred to a solution of Nfluorobenzenesulfonimide (30.6 g, 97.1 mmol) in 100 ml THF at 0°C. reaction see stirred for 15 min at 0°C and then at room temperature. during the night. reaction see evaporated, added EtOAc and reaction. cm. washed successively with water, 0.1 n. HCl solution, saturated NaHCO ₃ solution and brine. OL was dried over MgSO4, filtered and evaporated. The product was purified on silica gel, eluent: DCM^1% MeOH in DCM. Purified fractions were evaporated to give intermediate 13 (21.4 g, quantitatively).

Intermediate 14.

A solution of intermediate 13 (21.4 g, 77.728 mmol) was stirred in MeOH (500 mL) at rt. NaOH (486 ml, 2 M, 973 mmol) was added and the mixture was stirred for 2 hours at RT. reaction see acidified with 1 n. HCl and the product was extracted with Et ₂ O. OL was washed with saline and dried over MgSO ₄ , filtered and evaporated. The product was purified on silica gel, eluent: DCM ^ 5% MeOH in DCM. Purified fractions were evaporated to give 14 (15 g, 74%).

- 25 039788

Intermediate 15.

Intermediate 14 (15 g, 57.407 mmol) was dissolved in DCM (1000 ml). N,O-dimethylhydroxylamine hydrochloride (11.2 g, 114.8 mmol) and Et ₃ N (17.4 g, 172 mmol) were then added. The reaction mixture was cooled to 0°C. Then HATU (24.0 g, 63.1 mmol) was added. The reaction mixture was stirred at rt. within 2 hours Reaction. see poured into aq. NaHCO ₃ solution (100 ml). OL was separated, dried with MgSO ₄ and the solvent was evaporated. The residue was purified by silica gel flash column chromatography, eluent: DCM^1% MeOH in DCM. The product fractions were collected and the solvent was evaporated to give the desired product 15 (8.49 g, 49%).

Intermediate 16.

Intermediate 15 (8.49, 27.9 mmol) in THF (60 mL) was placed in a flask under N2 and cooled to -15°C. Methylmagnesium bromide (16.3 ml, 3 M, 48.8 mmol) was added dropwise while the temperature did not exceed 0°C. After adding react. cm. stirred for 1 h at rt. Then reaction. see poured onto ice with 20 ml AcOH. The product was extracted with Et ₂ O and OL washed with 5% NaHCO ₃ solution. OL was dried over MgSO ₄ , filtered and evaporated and purified on silica gel, eluent:

DCM. Purified fractions were evaporated to give intermediate 16 (3.20 g, 44%). Intermediate 17.

Intermediate 16 (3.2 g, 0.0123 mol) was stirred in toluene (150 ml) at 0° C. under N2. Potassium tert-butoxide (1.94 g, 17.3 mmol) was added at 0-5°C, ethyl difluoroacetate (1.84 g, 0.0149 mol) was added dropwise at 0-5°C. reaction see stirred at rt. within 2 hours Reaction. cm. washed with 10% H ₂ SO ₄ in water and OL dried over MgSO ₄ , filtered and evaporated to give intermediate 17 (4.16 g, 99%).

Intermediate 18.

Intermediate 17 (4.16 g, 12.3 mmol) and 1H-1,2,4-triazol-5-amine hydrochloride (2.97 g, 24.7 mmol) in DMF (40 ml) were stirred at 80° C for 16 hours Reaction. see evaporated, DCM was added and 2 g (Boc) ₂ O and 2 ml Et ₃ N were added.

The mixture was stirred for 30 min, washed with water, OL was dried over MgSO ₄ , filtered and evaporated. The product (4 isomers) was purified on silica gel, eluent: DCM^2% MeOH in DCM. Fractions were evaporated to give 3.55 g of the crude material, which was purified by preparative HPLC (stationary phase: Uptisphere® C18 ODB - 10 µm, 200 g, 5 cm, mobile phase: 0.25% NH4HCO3 solution in water , CH ₃ CN) to give intermediate 18 (730 mg, 15%).

Intermediate 19.

F

To intermediate 18 (0.73 g, 1.894 mmol) in MeOH (20 ml) was added HCl (6 M in iPrOH) (20 ml, 6 M, 120 mmol) and the resulting mixture was stirred at rt. during the night. Solvents were evaporated to give intermediate 19 (600 mg, 99%).

Alternative procedure for preparing intermediate 19 and separation into intermediates 19a and 19b.

intermediate connection intermediate connection intermediate connection

19a 19c

To intermediate 18 (4.5 g, 11.7 mmol) in DCM (52 ml) was added trifluoroacetic acid (TFA) (6 M in iPrOH) (5.4 ml, 6 M, 70 mmol) and the resulting mixture was stirred at r. .t. within 1 h the Solvents were evaporated and the resulting residue was dissolved in DCM and washed with saturated aqueous NaHCO ₃ . OL was separated and the aqueous layer was re-extracted with 2 x DCM. The combined OL was dried over MgSO ₄ , filtered and evaporated under reduced pressure. The resulting residue was purified by silica gel flash column chromatography using a DCM/NH ₃ (MeOH) gradient from 99/1 to 93/7 as eluent to give intermediate 19 (2.2 g, 66% yield) as racemic mixtures. This was separated into enantiomers by preparative SFC (stationary phase: Chiralpak Diacel AD 20x250 mm, mobile phase: CO ₂ , EtOH+0.4% iPrNH 2 ) to give intermediates 19a (955 mg, 29%, Rt=2.36 min ) and 19b (970 mg, 29%, R _t =2.99 min).

Intermediate 20.

8-(1,1-Dimethylethyl)-2-methyl ester of 8-azabicyclo[3.2.1]octane-2,8-dicarboxylic acid [1033820-28-8] (4.77 g, 17.71 mmol) was stirred in MeOH (41.608 ml) at rt. NaOH (106 mL, 1 M, 106 mmol) was added and the mixture was stirred overnight at rt. The MeOH was evaporated. reaction see acidified with 1 n. HCl and the product was extracted with chloroform. OL was dried over MgSO ₄ , filtered and evaporated to give intermediate 20 (4.52 g, 100%).

Intermediate 21.

Oh Oh

Intermediate 20 (4.52 g, 17.704 mmol) was dissolved in DCM (200 ml). N,O-dimethylhydroxylamine hydrochloride (3.454 g, 35.407 mmol) and Et ₃ N (5.37 g, 53.1 mmol) were then added. The reaction mixture was cooled to 0°C. Then HATU (7.41 g, 19.5 mmol) was added. The reaction mixture was stirred at rt. within 2 hours the Reaction mixture was poured into aq. NaHCO ₃ solution (100 ml). OL was separated, dried with MgSO ₄ and the solvent was evaporated. The residue was purified by silica gel flash column chromatography, eluent: DCM^1% MeOH in DCM.

The product fractions were collected and the solvent was evaporated to give intermediate 21 (3.03 g, 57%).

- 27 039788

Intermediate 22.

Intermediate 21 (3.03 g, 10.2 mmol) in THF (50 mL) was placed in a flask under N2 and cooled to -15°C. Methyl magnesium bromide (12.7 ml, 1.4 M, 17.8 mmol) was added dropwise while the temperature did not exceed 0°C. After adding react. cm. stirred for 1 h at rt. Then reaction. see poured onto ice with AcOH (20 ml). The product was extracted with Et ₂ O and OL washed with 5% NaHCO ₃ solution. OL was dried over MgSO ₄ , filtered and evaporated and purified on silica gel, eluent: DCM. Purified fractions were evaporated to give intermediate 22 (2.57 g, 100%).

Intermediate 23.

Intermediate 22 (2.57 g, 0.0101 mol) was stirred in toluene (150 ml) at 0° C. under N2. Potassium tert-butoxide (1.59 g, 14.2 mmol) was added at 0-5°C, ethyl difluoroacetate (1.52 g, 0.0122 mol) was added dropwise at 0-5°C. reaction see stirred at rt. within 2 hours Reaction. cm. washed with 10% H ₂ SO ₄ in water and OL dried over MgSO ₄ , filtered and evaporated to give intermediate 23 (3.34 g, 99%).

Intermediate 24.

Intermediate 23 (3.34 g, 10.1 mmol) and 1H-1,2,4-triazol-5-amine hydrochloride (2.43 g, 20.2 mmol) in DMF (30 ml) were stirred at 80° C for 16 hours Reaction. see evaporated, DCM was added and 2 g of (Boc) ₂ O and Et ₃ N (2 ml) were added. The mixture was stirred for 30 min, washed with water, OL was dried over MgSO ₄ , filtered and evaporated. The product (4 isomers) was purified on silica gel, eluent: DCM^2% MeOH in DCM. Fractions were evaporated to give 3.07 g of the crude material, which was purified by preparative HPLC (stationary phase: Uptisphere® C18 ODB - 10 μm, 200 g, ID 5 cm, mobile phase: 0.25% solution of NH ₄ HCO ₃ in water , MeOH) to give intermediate 24 (1.07 g, 28%).

Intermediate 25.

To intermediate 24 (1.07 g, 2.82 mmol) in MeOH (30 ml) was added HCl (6 M in iPrOH, 30 ml, 6 M, 179 mmol) and the reaction mixture was stirred at rt. during the night. The solvents were evaporated and the product was crystallized from ether. The crystals were filtered off and dried to give intermediate 25 as hydrochloric acid salt (1.12 g, 112%).

Alternative procedure for preparing intermediate 25.

Stage 1 Intermediate 43.

n-Butyl lithium (nBuLi, 106.5 ml, 266.3 mmol, 2.5 M in hexanes) was added at 0° C. to a solution of diisopropylamine (26.95 g, 266.3 mmol) in THF (500 ml). The reaction mixture was stirred at 0°C for 30 min, then it was cooled to -78°C and treated with a solution of N-Boc-nortropinone (50 g, 221.9 mmol) in THF (75 ml). The resulting mixture was stirred at -78°C for 90 min, then methyl cyanoformate (22.9 ml, 288.5 mmol) was added. The reaction was heated. see to k.t. and mixed it into

- 28 039788 during the night.

The reaction mixture was quenched with saturated aqueous NH4Cl then diluted with EtOAc. The organic layer was separated, then washed with water and brine, dried over MgSO ₄ , filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel flash column chromatography using a gradient of 100% DCM to 1% MeOH in DCM as eluent to give Intermediate 43 (60.25 g, 95.8%).

Stage 2 Intermediate 44.

Sodium borohydride (16.02 g, 423.5 mmol) was added to a cold (ice bath) solution of intermediate 43 (60 g, 211.8 mmol) in methanol (700 ml) and the reaction mixture was allowed to stir for 5 h. the reaction was quenched with saturated aqueous NH ₄ Cl, then the solvent was evaporated to dryness under reduced pressure. The resulting residue was dissolved in water and extracted with 3 x DCM. The combined organic layers were dried over MgSO ₄ , filtered and concentrated in vacuo to give intermediate 44 (59 g, 97.6%) which was used as such without further purification.

Stage 3 Intermediate 45.

Trifluoroacetic anhydride (57.5 mL, 413.5 mmol) was very slowly added dropwise (WARNING! exothermic reaction) to a solution of intermediate 44 (59 g, 206.8 mmol), triethylamine (115 mL, 827.1 mmol) and 4-(dimethylamino)pyridine (2.52 g, 20.7 mmol) in DCM (800 ml), keeping the temperature below 60°C. The reaction mixture was stirred at rt. overnight, then it was cooled in an ice bath and quenched with water.

The pH of the resulting solution was adjusted to 8-8.5 using a saturated aqueous solution of NaHCO ₃ and it was stirred at rt. for 1 hour Then the aqueous layer was extracted with 3 x DCM, then the combined organic layers were dried over MgSO ₄ , filtered and concentrated in vacuo. The resulting residue was purified by silica gel flash column chromatography using a gradient of 100% DCM to 2% MeOH in DCM as eluent to give Intermediate 45 (33.7 g, 61%).

Stage 4 Intermediate 46.

MeOH (59 ml) was added to a flask containing intermediate 45 (2.44 g, 9.13 mmol) and Pd/C (10%) (0.971 g, 0.913 mmol) under nitrogen atmosphere. The resulting vacuum was created and filled with gaseous hydrogen, and it was stirred at rt. during the night. The reaction mixture was filtered through a pad of Celite®, then the solvent was removed in vacuo. The resulting residue was purified by silica gel flash column chromatography using a gradient of 100% DCM to 2% MeOH in DCM as eluent to give Intermediate 46 (2 g, 81%).

Step 5 Intermediate 47

LDA (54.3 ml, 108.7 mmol, 2 M in cyclohexane/ethylbenzene/THF) was added dropwise at -78°C to -60°C to a solution of intermediate 46 (24.4 g, 90. 6 mmol) in anhydrous THF (363 ml) under nitrogen atmosphere. After the addition, the reaction mixture was heated to -30°C and stirred for 10 min, after which it was cooled back to -78°C. Dissolve 7-chloro-5(difluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidine ([1340394-63-9], 18.5 g, 90.6 mmol) in a minimum amount of THF and added dropwise with a syringe. The reaction mixture was stirred at -60°C for 1 h, then the temperature was raised to rt. and the reaction mixture was quenched with saturated aqueous NH ₄ Cl. The volatiles were evaporated in vacuo and the resulting residue was partitioned between EtOAc and

- 29 039788 with water. The layers were separated, then the aqueous layer was extracted with 2 x EtOAc. The combined OL was washed with brine, dried over MgSO ₄ , filtered and concentrated under reduced pressure. The resulting residue was crystallized from diethyl ether to give intermediate 47 (23.8 g, 60%). The filtrate was evaporated and the resulting residue was purified by silica gel flash column chromatography using a gradient of 100% DCM to 2% MeOH in DCM as eluent to give intermediate 47 (2 g, 5%) after recrystallization from diethyl ether.

Step 6 Intermediate 25, Intermediate 25a and Intermediate 25b.

Stir intermediate 47 (25.8 g, 58.9 mmol) in concentrated HCl solution (266 ml, 3.19 mol, 37% in H ₂ O) at 150° C. overnight. The volatiles were evaporated in vacuo and the resulting residue was co-evaporated twice with toluene. The product was treated with saturated aqueous NaHCO ₃ until basic pH was reached. The aqueous layer was extracted with DCM, then the organic layer was dried over MgSO ₄ , filtered and evaporated under reduced pressure. The resulting residue was purified by silica gel flash column chromatography using a gradient of 100% DCM to 8% MeOH/NH ₃ in DCM as eluent to give Intermediate 25 as a racemic mixture (13 g, 79%). This material was purified by preparative SFC (stationary phase: Chiralpak Diacel AD 20x250 mm, mobile phase: CO ₂ , EtOH+0.4 iPrNH 2 ). Convert the 1S,4S,5S enantiomer to its HCl salt and recrystallize from CH3CN to give intermediate 25a (5.58 g, 30%, R _t = 2.91 min, [a] ²⁰ _D = -66.6 (s =0.48, DMF)).

Intermediate 26.

A mixture of 1-tert-butyl-3-methyl-4-(((trifluoromethyl)sulfonyl)oxy)-5,6-dihydropyridine-1,3(2H)-dicarboxylate [161491-25-4] (15 g, 38 .53 mmol, prepared according to Angew Chem Int Ed 2015, 54, 12942-12946), N-butylboronic acid [4426-47-5] (5.89 g, 57.8 mmol), Pd( PPh ₃ ) ₄ [14221-01-3] (1.78 g, 1.54 mmol) and Na ₂ CO ₃ [497-19-8] (8.167 g, 77.052 mmol) in 1,4-dioxane [123- 91-1] (300 ml) and heated at 90°C overnight. The volatiles were evaporated in vacuo and the resulting residue was treated with 1N HCl. aqueous solution of HCl. The aqueous layer was extracted with DCM, then the combined OL was dried over MgSO ₄ , filtered and evaporated in vacuo. The resulting residue was purified by silica gel flash column chromatography using a DCM-MeOH (9:1, o6./o6.)/DCM 0/100 to 30/70 gradient as eluent). 3.65 g of 1-tert-butyl-5-methyl-4-butyl-3,6-dihydro-2H-pyridine-1,5-dicarboxylate was thus obtained, which was dissolved in a mixture of methanol and 1 N hydrochloric acid. aqueous NaOH solution (1:1, v/v, 200 ml) and stirred overnight at rt. The volatiles were evaporated under reduced pressure, and the resulting residue was placed on ice and acidified with 1N HCl. aqueous solution of HCl. The aqueous layer was extracted with chloroform, then the combined OLs were dried over MgSO4, filtered and evaporated under reduced pressure. The resulting residue was purified by silica gel flash column chromatography using a gradient of 1% MeOH in DCM to 2% MeOH in DCM as eluent to give Intermediate 26 (1.69 g, 15.5% yield).

Intermediate 27.

- 30 039788

Intermediate 26 (1.47 g, 5.188 mmol) was added to a suspension of Pd/C (10%) (668 mg,

0.63 mmol) in MeOH [67-56-1] (134 ml). The mixture was then placed under an H ₂ atmosphere and stirred for 36 hours. At the end of this time, the catalyst was filtered through a pad of Celite and the solvent was evaporated under reduced pressure to give intermediate 27 (1.45 g, 98%).

Intermediate 28.

Following a similar procedure to that given for the synthesis of Intermediate 8, Intermediate 28 was synthesized from Intermediate 27 to give Intermediate 28 (200 mg).

Intermediates 29, 29a and 29b.

NaH (60% dispersion in mineral oil, 1.5 g, 39.05 mmol) was added to a solution of tert-butyl-3[5-(difluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidine -7-yl]piperidine-1-carboxylate which was prepared analogously to intermediate 8 and 11 starting from 3-pyridinecarboxylic acid (11.5 g, 32.54 mmol) in DMF (500 ml) at rt. and in stream N ₂ . The reaction mixture was stirred for 30 min at rt, then methyl iodide (5.54 g, 39.05 mmol) was added dropwise and the mixture was stirred overnight at rt. Water was added and the product was extracted with EtOAc. OL was washed with brine, dried over MgSO ₄ , filtered and evaporated. The resulting residue was purified by preparative HPLC (stationary phase: Uptisphere® C18 ODB - 10 μm, 200 g, ID 5 cm, mobile phase: 0.25% NH ₄ HCO ₃ solution in water, MeOH) to give intermediate 29 (4 .32 g, 36%) as a racemic mixture. This was separated into enantiomers by preparative SFC (stationary phase: Chiralcel Diacel OJ 20x250 mm), mobile phase: CO ₂ , iPrOH+0.2% iPrNH ₂ ) to give intermediates 29a (2 g, 17%) and 29b (2 d, 17%).

Intermediate 30a.

To intermediate 29a (500 mg, 1.36 mmol) in MeOH (20 ml) was added HCl (6 M in iPrOH, 20 ml, 6 M, 120 mmol) and the reaction mixture was stirred at rt. during the night. Solvents were evaporated to give intermediate 30a as hydrochloric acid salt (400 mg, 97%).

Intermediate 30b.

Intermediate 30b was prepared in the same manner as described for intermediate 30a starting from 29b (400 mg, 97%).

- 31 039788

Alternative procedure for preparing intermediate 29. Step 1. Intermediate 31.

K ₂ CO ₃ (33.15 g, 0.24 mol) was added to a stirred solution of 1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid [84358-12-3] (50.0 g, 0.22 mol) in DMF (600 ml) at rt. After 30 min, methyl iodide (34.05 g, 0.24 mol) was added and the reaction mixture was stirred for an additional 2.5 h at rt. The volatiles were evaporated in vacuo and the resulting residue was taken up in water and extracted with DIPE. The combined OL was dried over MgSO ₄ , filtered and evaporated to give 54.75 g of intermediate 31, which was used as such in the next step.

Stage 2 Intermediate 32.

LiHMDS (450 ml, 0.45 mol, 1 M) was added dropwise to a solution of intermediate 31 (54.75 g, 0.22 mmol) in THF (1 L) at -78°C and under N2 atmosphere. The solution was stirred at -78° C. for 1 h and then methyl iodide (63.9 g, 0.45 mmol) was added dropwise at this temperature. After completion of the addition, the reaction mixture was heated to rt. Then reaction. see treated with saturated aqueous NH ₄ Cl. The aqueous layer was extracted with Et ₂ O. The combined OL was dried over MgSO 4 , filtered and evaporated. The resulting residue was purified by flash column chromatography using 20% EtOAc in heptane as eluent to give Intermediate 32 (55.5 g, 96% yield).

Intermediate 29.

Following a synthetic procedure similar to that given for the synthesis of Intermediate 8, starting from Intermediate 32, Intermediate 29 (36.7 g, 3.5% yield 4) was obtained.

Intermediate 33.

NaH (60% dispersion in mineral oil, 0.97 g, 24.2 mmol) was added to intermediate 6 (5 g, 20.7 mmol) in anhydrous THF (35 ml), in 250 ml four-necked RBF, at 0° C in stream N2. After 10 minutes, carbon disulphide (1.46 ml, 24.1 mmol) was added dropwise and then methyl iodide (2.71 ml, 43.5 mmol) was added dropwise. The mixture was stirred overnight at rt. Water was added and the product was extracted with EtOAc. OL was washed with brine, dried over MgSO4, filtered and evaporated to give intermediate 33 (8.2 g, 92%) which was used as such in the next step.

- 32 039788

Intermediate 34.

Intermediate 33 (1 g, 2.89 mmol) and 1H-1,2,4-triazol-5-amine hydrochloride (0.35 g, 2.89 mmol) were stirred at 150°C for 3 h to proceed with the reaction melting. reaction see cooled to rt and dissolved in DCM. OL was washed with sat. aq. sol. NaHCO ₃ , water, brine, dried over MgSO4, filtered and evaporated. The product was purified on silica gel, eluent DCM/MeOH 100/0 to 98/2, to give intermediate 34 (200 mg, 26%).

Intermediate 35.

To Intermediate 34 (200 mg, 0.76 mmol) in DCM (3.3 ml) was added (Boc) ₂ O (199 mg, 0.91 mmol), Et ₃ N (127 μl, 0.91 mmol) and reaction see stirred at rt. during the night. The solvent was evaporated, then the residue was taken up in DCM and washed with water (3x), then brine. OL was separated, dried (MgSO ₄ ), filtered and evaporated to give intermediate 35 (199 mg, 72%) which was used without further purification in the next step.

Intermediate 36.

To intermediate 35 (347 mg, 0.95 mmol) in CCl ₃ (12 mL) was added mCPBA (659 mg, 3.82 mmol) and react. cm. heated under reflux for 2 hours. see diluted with CHCl ₃ , washed with 1 n. NaOH, dried over MgSO ₄ , filtered and evaporated to give intermediate 36 (301 mg, 79%), which was used as such in the next step.

Intermediate 37.

Intermediate 36 (799 mg, 2.02 mmol) and sodium cyanide (202 mg, 4.04 mmol) were stirred in DMSO (5 mL) at rt. within 30 min. reaction cm. was poured into water and extracted with EtOAc. OL was washed with brine, dried over MgSO ₄ , filtered and evaporated to give intermediate 37 (550 mg, 80%), which was used as such in the next step.

Intermediate 38.

TFA (6 ml) was added to intermediate 37 (520 mg, 1.52 mmol) in DCM (3.0 ml) and react. see stirred for 1 h at 0°C. reaction see poured into a saturated solution of Na ₂ CO ₃ , then

- 33 039788

OL was washed again with saturated Na ₂ CO ₃ , brine, dried over MgSO ₄ , filtered and evaporated to give intermediate 38 (273 mg, 73%).

Intermediate 39.

Intermediate 6' (prepared from intermediate 31 according to the same transformations as for intermediates 3-6) (50 g, 0.22) was added to N,N-dimethylformamide dimethyl acetal (110 mL) and the mixture was refluxed for 4 days. The reaction mixture was evaporated and additional N,N-dimethylformamide dimethyl acetal was added and the mixture was heated at reflux for an additional 4 hours. The solvent was evaporated and the product was purified on silica gel, eluent: 1% MeOH in DCM, 2%. Purified fractions were evaporated to give intermediate 39 (60 g, 97%).

Intermediate 40.

A solution of intermediate 39 (60 g, 0.21 mol) and 1H-1,2,4-triazol-5-amine hydrochloride (22.3 g, 0.27 mol) in acetic acid (53 ml) was stirred with heating with reflux for 1 hour. Water was added and the product was extracted with ether. OL was washed with brine and dried over MgSO ₄ , filtered and evaporated to give intermediate 40 (62 g, 96%).

Intermediate 41.

Intermediate 40 (500 mg, 1.65 mmol) was stirred in DMF (50 mL) at rt. in stream N2. NaH (60% dispersion in mineral oil, 72 mg, 1.8 mmol) was added and the mixture was stirred for 30 min. Methyl iodide (2.57 mg, 1.8 mmol) was added and the mixture was stirred for 2 h at rt. The reaction mixture was quenched with water and evaporated. Water was added and the product was extracted with EtOAc. OL was washed with brine, dried over MgSO ₄ , filtered and evaporated. This was purified by preparative HPLC on (RP Vydac Denali C18-10 µm, 200 g, 5 cm; mobile phase (0.25% NH4HCO3 solution in water, MeOH) to give Intermediate 41 (250 mg, 48%).

Intermediate 42.

Intermediate 41 (200 mg, 0.85 mmol) was stirred in MeOH (20 ml) and 6N was added. HCl in i-PrOH (23 ml). The mixture was stirred for 1 hour. React. cm. evaporated to give intermediate 42 (250 mg, 100%).

Intermediate 48.

- 34 039788

Stage 1 Intermediate 49.

O^O

X JTX

NaH (1.37 g, 34.4 mmol, 60% dispersion in mineral oil) was added to a suspension of methyl 2hydroxy-6-methylisonicotinate (2.3 g, 13.76 mmol) in ACN (150 ml) at 0°C and in an N2 atmosphere. Ensured the achievement of reactions. see k.t. and stirred for 45 min. Then 2,2difluoro-2-(fluorosulfonyl)acetic acid (3.16 g, 17.76 mmol) was added dropwise to the reaction mixture, which was further stirred at rt. within 15 hours. At the end of this time, it was quenched with saturated aqueous NH4Cl and the resulting mixture was extracted with DCM (100 ml x3). The combined organic extracts were dried over MgSO ₄ , filtered and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography using a 100/0 to 50/50 heptane/EtOAc gradient as eluent to give Intermediate 49 (2.21 g, 73.9%).

Stage 2 Intermediate 48.

An aqueous solution of NaOH (15 ml, 15 mmol, 1 M) was added to a solution of intermediate 49 (2.11 g, 9.72 mmol) in ethanol (15 ml). The resulting mixture was stirred for 1 h, then it was treated with an aqueous solution of HCl (15 ml, 15 mmol, 1N). A white precipitate formed and was filtered, washed with cold water, then dried in a vacuum oven overnight to give Intermediate 48 (1.75 g, 88.6%).

Intermediate 50.

0^0 AND

Stage 1 Intermediate 51.

Concentrated H2SO4 (0.791 ml, 4.85 mmol, 1.84 g/ml) was added to a solution of 2,6-dichloro-3-fluoroisonicotinic acid ([149468-00-8], 5.5 g, 26.19 mmol) in MeOH (99 ml) and the resulting mixture was heated to 85°C for 12 hours. After this time, additional H2SO4 (0.791 ml, 14.846 mmol, 1.84 g/ml) was added and the reaction. see stirred at 90°C for 24 hours the Reaction mixture was cooled to rt, evaporated the solvent and added ice to the resulting residue. A precipitate formed and was filtered, washed with ice water and dried in a vacuum oven for 2 days to give Intermediate 51 (4.35 g, 19.4 mmol) as a beige solid.

Step 2 Intermediates 52a and 52b.

Intermediate 52a Intermediate 52b

A solution of intermediate 51 (3.95 g, 17.6 mmol) and trimethylboroxine (1.66 ml, 5.82 mmol, 3.5 M in THF) in anhydrous 1,4-dioxane (128 ml) was degassed for 15 min. Palladium(II) acetate (198 mg, 0.88 mmol), tricyclophenylphosphine (494 mg, 1.76 mmol) and tribasic potassium phosphate (11.23 g, 53.0 mmol) were added successively and the resulting mixture was degassed, then stirred and heated at 110° C. for 18 hours in a pressure reaction tube. reaction see cooled to rt and evaporated the solvent in vacuo. The resulting residue was partitioned between water and EtOAc. The resulting biphasic mixture was separated and the aqueous layer was extracted with EtOAc (3x). The combined organic layers were washed with brine (1 x), dried over MgSO ₄ , filtered and the solvent was evaporated in vacuo. The resulting residue was purified by silica gel flash column chromatography using a 100/0 to 90/10 heptane/EtOAc gradient as eluent to give intermediate 52a (980 mg, 27%) and its regioisomer 52b (460 mg, 12.8% ).

- 35 039788

Step 3 Intermediate 50

To a stirred solution of intermediate 52a (785 mg, 3.85 mmol) in THF (10 mL) was added a solution of LiOH (277 mg, 11.5 mmol) in water (10 mL). reaction see stirred at room temperature for 1.5 h, then the volatiles were evaporated in vacuo. The pH of the resulting aqueous residue was adjusted to ~2 by treatment with an aqueous solution of HCl (1 M). A white precipitate formed and was filtered, washed with ice water and dried in a vacuum oven for 2 days to give Intermediate 50 (491 mg, 67%) as a white solid.

Intermediate 53.

To a cold (0°C) solution of methyl 5-fluoro-2-methoxyisonicotinate (4 g, 21.6 mmol) in DCM (71 ml) was added methyltrioxorhenium (TP) (0.443 g, 1.78 mmol) in one portion followed by dropwise addition of hydrogen peroxide (64.3 ml, 0.735 mol, 35% in water). reaction see left to heat up to rt. and then heated to 35°C. After 24 h, additional methyltrioxorenium (TP) (0.2 g, 0.80 mmol) was added and react. see left to mix at 35°C for 2 days.

Portions, 3x over 8 h, were added methyltrioxorhenium (TP) (0.2 g, 0.802 mmol) and hydrogen peroxide (30 ml, 0.343 mol, 35% in water) and react. see stirred at 27°C for 16 hours Reakt. cm. was cooled in an ice bath, then it was quenched by adding portions of manganese dioxide (WARNING! strong evolution of gas and heat) until the evolution of gas ceased to obtain a black mixture. This was combined with another batch of the reaction mixture resulting from a reaction starting with 1 g of methyl 5-fluoro-2-methoxyisonicotinate. The combined reactions were filtered through a Celite® plug, which was washed with DCM. The biphasic filtrate was separated and the aqueous phase was extracted with DCM (3x20 ml). The combined organic layers were dried over MgSO ₄ , filtered and evaporated in vacuo. The resulting residue (4.9 g) was purified by silica gel flash column chromatography using a 100/0 to 0/100 heptane/EtOAc gradient as eluent to give intermediate 54 (585 mg).

Stage 2 Intermediate 55.

A suspension of intermediate 54 (585 mg, 2.91 mmol) in phosphorus oxychloride (4.1 ml, 43.6 mmol) was stirred and heated to 105° C. in an oil bath. After 1.5 h the solvent was removed in vacuo. Ice (10 ml) was added to the resulting residue and the aqueous mixture was extracted with DCM (3x20 ml). The combined organic layers were washed with brine, dried over MgSO ₄ and evaporated in vacuo. The resulting residue (580 mg) was purified by silica gel column chromatography using a 100/0 to 50/50 heptane/DCM gradient as eluent to give Intermediate 55 (522 mg, 72.7%).

Stage 3 Intermediate 53.

A solution of LiOH (152 mg mg, 6.35 mmol) in water (5.7 ml) was added to a stirred solution of intermediate 55 (522 mg, 2.12 mmol) in THF (5.7 ml). reaction see stirred at room temperature for 1.5 h, then the volatiles were evaporated in vacuo. The pH of the resulting aqueous residue was adjusted to ~3 by treatment with an aqueous solution of HCl (1 M). A white precipitate formed and was filtered, washed with ice water and dried in a vacuum oven to give Intermediate 53 (172 mg, 39%) as a white solid. The filtrate was evaporated to dryness under reduced pressure to give the lithium salt of compound 53 (366 mg).

- 36 039788

B. Synthesis of final compounds. Compound 1a and compound 1b.

Comm. No. 1a Conn. No. 1b

A mixture of I-12 (0.27 g, 0.001 mol) and 2,6-dimethylisonicotinic acid (0.16 g, 0.001 mol) in DCM (10 mL) was treated with DIPEA (0.69 mL, 0.75 g/m). ml, 0.004 mol) and HBTU (0.38 g, 0.001 mol). Stirring was continued for 16 hours. see diluted with water (5 ml), acidified with 1 M HCl to pH~3, and the layers were separated, and OL washed with 1 M NaOH until pH~9, water, then dried over MgSO ₄ , filtered and concentrated in vacuo to give an oil (0.8 g). Purification was performed by preparative HPLC (stationary phase: RP XBridge® Prep C18 OBD10 µm, 50x150 mm, mobile phase: 0.25% NH4HCO3 solution in water, MeOH) to obtain two fractions. Purification was performed using Prep SFC (stationary phase: Chiralpak® Diacel AD 20x250 mm, mobile phase: CO ₂ , EtOH with 0.4% iPrNH 2 ) to give 4 fractions, two of which provided compound 1b (64 mg, 18%) and 1a (70 mg, 19%).

Compounds 2a-3b were prepared in the same way as compounds 1a and 1b from the indicated starting material.

1-12a

Connection 4.

2-Methyl-6-(trifluoromethyl)isonicotinic acid (101 mg, 0.493 mmol) was stirred in DCM (20 ml). DIPEA (0.34 ml, 0.75 g/ml, 1.97 mmol) and HBTU (206 mg, 0.542 mmol) were added and stirring was continued for 20 minutes at rt. I-13a (150 mg, 0.493 mmol) was added and stirring continued overnight at rt. reaction cm. extinguished with water, stirred for 20 min, then separated OL. The aqueous layer was re-extracted with 2 x DCM. The combined organic layers were washed with brine, then separated and dried over MgSO4, filtered and concentrated in vacuo. This material was purified by silica gel flash chromatography eluting with 0 to 5% MeOH in DCM to give crude compound 4 (120 mg, 52.931% yield). Purification was performed by preparative HPLC (stationary phase: RP Vydac Denali C18-10 µm, 200 g, I.D. 5 cm, mobile phase: 0.25% NH4HCO3 solution in water, MeOH) to give after evaporation with

MeOH and drying in a vacuum oven overnight compound 4 (74 mg, 36%).

Compounds 5-23 were prepared similarly to compound 6 using enantiomerically pure intermediate I-13a.

conn. no. Structure Yield (%)

- 38 039788

- 39 039788

Compound 24a and compound 24b.

HBTU (0.28 g, 0.00075 mol) and DIPEA (0.54 ml, 0.75 g/ml, 0.0031 mol). After stirring the mixture for 30 minutes, I-19 (0.2 g, 0.00062 mol) was added in one portion. reaction see left to stir for 1 h and then added 1 M NaOH (5 ml), the layers were separated and OL was washed with water (10 ml), dried over MgSO ₄ , filtered and concentrated in vacuo to obtain an oil. This was purified by flash chromatography using a 24 g Redisep® flash column eluting with a gradient of 0 to 5% MeOH in DCM to give an oil which was crystallized from DIPE (10 ml) to give a white solid (0 .23 g, 83.2% yield. Purification was carried out with preparative SFC (stationary phase: Chiralpak® Diacel AD 30x250 mm, mobile phase: CO ₂ , iPrOH with 0.2% iPrNH 2 ) to obtain two fractions. Both fractions were transferred into test tubes and dried in a vacuum oven (45° C., 16 hours) thus obtaining amorphous solids comp. No. 24a (0.11 g, 39.7% yield) and comp. No. 24b (0.11 g, 38.3% yield).

Compounds 25a and 25b.

Comm. No. 25a Conn. No. 25b

Benzothiazole-6-carboxylic acid (142 mg, 0.792 mmol) was stirred in DCM (15 ml), DIPEA (0.82 ml, 4.8 mmol) and HBTU (300 mg, 0.792 mmol) were added. Stirring was continued for 0.5 h at rt. Intermediate 25 (250 mg, 0.792 mmol) was added to the solution and stirring was continued for 2 h at rt. NaOH solution (1N, 1 ml) was added and stirred for 5 minutes. The product was filtered through an Extrelute filter and the filtrate was evaporated. The product was purified on silica gel, eluent: DCM^4% MeOH in DCM. Purified fractions were evaporated to give a mixture of compounds 25a and 25b (340 mg). This was purified with preparative SFC (stationary phase: Chiralcel® Diacel OD 20x250 mm, mobile phase: CO ₂ , EtOH+0.4 iPrNH ₂ ) to give two products, both of which were crystallized from Et ₂ O to give comp. No. 25a (121 mg, 35%) and comp. No. 25b (128 mg, 37%).

- 40 039788

Compounds 26a and 26b.

about

Comm. No. 26a

Comm. No. 26b

2,6-Dimethylisonicotinic acid (154.845 mg, 1.0 mmol) was stirred in DCM (10 ml). DIPEA (0.53 ml, 3.1 mmol) and HBTU (427 mg, 1.1 mmol) were added, stirring was continued for 0.5 h at rt. Intermediate 38 (273 mg, 1.1 mmol) was dissolved in DCM (5 ml) and this mixture was added to the solution, with stirring continued for 3.5 h at room temperature. reaction see quenched with water, then the two layers were separated and aq. sl. re-extracted with DCM. OL was dried over MgSO ₄ , filtered and evaporated. The product was purified on silica gel, eluent: DCM/MeOH from 100/0 to 97/3 to 94/6, which provided two pairs of diastereomers as a mixture. Purification was carried out using preparative SFC (stationary phase: Chiralcel® Diacel OD 20x250 mm, mobile phase: CO ₂ , EtOH+0.4 iPrNH ₂ ), which resulted in two trans-enantiomers. Purification was performed by preparative HPLC (stationary phase: RP XBridge Prep C18 ODB - 5 μm, 30x250 mm, mobile phase: 0.25% solution of NH ₄ HCO ₃ in water, CH ₃ CN) to give compound 26a (26 mg, yield 6.761%) and 26b (20 mg, 5.201% yield).

Connection 321.

Intermediate 9b (.2HCl, 1.2 g, 3.527 mmol), 2-(difluoromethyl)-6-methyl-4-pyridinecarboxylic acid (660 mg, 3.527 mmol), EDCI (1.352 g, 7.053 mmol) and DIPEA (1.823 g, 14.107 mmol) in DCM (71.4 mL) was stirred at rt. within 4 hours Reaction. see washed with 1 n. NaOH, OL was dried over MgSO ₄ , filtered and evaporated. The product was purified on silica gel, eluent MeOH/DCM from 0/100 to 3/97. Purified fractions were evaporated and crystallized from DIPE. The crystals were filtered off and dried to give compound 321 (920 mg, 60%).

Connection 333.

To a stirred solution of intermediate 25a (100 mg, 0.317 mmol) in dry ACN (5 ml) was added 3,5-dichlorobenzoic acid (72.593 mg, 0.38 mmol). TEA (0.22 ml, 1.58 mmol) was added followed by 1-propanephosphonic anhydride ([68957-94-8], 0.28 ml, 0.48 mmol) and the mixture was stirred at rt. for 3 hours to give a white precipitate in a brown solution. The solvent was removed in vacuo and the crude material was partitioned between DCM (10 ml) and saturated aqueous NaHCO3 (10 ml). OL was washed with saturated aqueous Na ₂ CO ₃ (1x15 ml), dried (MgSO ₄ ), filtered, solvent removed in vacuo and co-evaporated with toluene (1x20 ml) to give a white precipitate in a brown solution which was dried overnight to give brown and white crystals. Recrystallization from Et ₂ O gave off white crystals which were oven dried overnight in a vacuum oven at 40° C. to give compound 333 (88 mg, 61%) as off white crystals.

In table. 1 below lists additional compounds that were obtained by analogy with the above examples. If the salt form is not indicated, the compound was obtained as the free base. conn. no. means connection number. The reagents used in the synthesis of the compounds were either commercially available or were obtained by procedures known to those skilled in the art. The reagent column lists compounds prepared in analogy to compound 321 (note: coupling by EDCI); the compounds obtained by analogy with compound 333 are indicated (note: phosphonic anhydride).

Table 1

No. conn. Structure Derived from Output (%) 27 CI l\L N O 1 x > N-U I-9b 78 28 hi N O ^V N-/ 1-25 35 29 Ϊ I ^x ? ¹ Λϊ N. .N О X // ^N N-if 1-25 39 thirty w&A N. N O // ^N N-Y 1-25 38 31 i G? ¹ ifS XN О ,Ν N-У Racemic, endo-isomer 1-25 26 32 F A---S fi N AVAV X N 0 1 n!/ I-9b 55 33 1 ΙίΑ') N.N.O Ύ,ν N-i/ 1-19 44 34 F XXX XXN । fj ' π ϊ N.N.O 1-19 41 35 JL JL J! A ^F Tl o I-9b 68 36 Α^θνΦ NL JM O F Ύ ;n N-U I-9b 73

- 42 039788

37 ° ^F v N-U I-12a 52 38 g “H/X NH ₂ 1 I 1 f| Ί ^F η 'ί π τ N. JJ O O I-9b 41 39 Cl N. Ν Ο CF ₃ χ ;ν ³ NV I-9b 21 40 N. N 0 X > N—V I-9b 78 41 F (Αν VAW 14 N Ο CF ₃ U ¹ I-9b 38 42 CI F ί|^ ^Ν 14 -Ν Ο χ ;.ν NV I-9b 60 43 CI Ww N. .Ν Ο CI χ ;ν Ν-Ι/ I-9b 55 44 L ^rs * 1 Τ RS Π N. -Ν Ο χ ;,ν Ν-ί/ Mixture of trans-isomers 1-28 29

- 43 039788

45 Lru 1 II S* Π O x ;n N-if Pure trans enantiomer 1-28 12 46 ΧΦΆ N. -ΝΟ X > N-V Pure trans enantiomer 1-28 12 47 VeyP I-9b 44 48 ARUYA* ^s I-9b 77 49 ϊ 'Ό i|V ^F Xu π NL Ν O I-9b 62 fifty X X pX> l\L N O o-if X ;.N \ N-if I-9b 68 51 F ------r^N hl -Ν O Cl X ^N I-9b 31 52 CF ₃ lcm N. .N 0 ¹ 1/ I-9b 6

- 44 039788

53 OMe N. N 0 ¹ I/ I-9b 17 54 Cl ^F (Y ^N N. -N OF x ;n NV I-9b 53 55 Cl f ^F y ^ N N. -N O x > N-Y I-9b 63 56 ^{F ρΧ 5ί Χΐΐ} τ — N. -N O x NZ< I-9b 51 57 F ''-g----S G a jC ^{n F} N. _N O' b ^m I-9b 61 58 / F -X^s ^-N hL N O Y ^N I-9b 75 59 .+xmY N.0 I? I-9b 72 60 ΐ P (|L ^F II 'l P 1 N. -N O I x ;.n NV I-9b 64 61 H F ^-N p^-Ox^xJQ ^{n F} H 'n P \ N. _N O >— Ύ,ν ^z NV I-9b 73

- 45 039788

62 N. .Ν Ο F 1-25 42 63 l H: y— Ζ ⁷ y—ζ I-9b 40 64 Cl _F Yes' l | 1 H L ^F H 4 Π Ν. Ή О 1/ I-9b 60 65 aDOd^ N. Ν θ' 1/ I-9b 49 66 N. JM Ο one/ I-9b 57 67 F -Ο r=^ rD/AZhD^- ^F Τι 'ί Π Ν. .Ν 0 b~ I-9b 63 68 ; ΥΊ ιΠ A^Q^/A N. Ν OF χ ;.ν N-V I-9b 48 69 η3~ ΛΆ Z—' Η C) I-9b 78 70 F ίί*^ ^Ν Α^^ΑνίίΑ ^Ν - Μ OF NV I-9b 58

- 46 039788

71 Cl ^F 'k3 A 1 JL j< JI kL ΊΐΓ ^cl Ν. -Ν O F x ;n NV I-9b 73 72 i Ό iY n! ή ο ^v kJ ¹ I-9b 38 73 LV Ύ>° I-9b 39 74 I 1 F Az° N. Ν O Ύ/Ν Ν-σ 1-19 thirty 75 eleven F A< N. .Ν About Ύ ,Ν Ν—V 1-19 32 76 F XXX AZ' ,Α^^ψίΧ N. .Ν Ο Ύ,ν N-U I-9b 36 77 eleven N. _Ν Ο Λί ,Ν N-U I-9b 42 78 LUJ N. Ν Ο one/ I-9b 58 79 one Gd, F^Y^r E1 ° N. Ν O one/ I-9b 74

- 47 039788

80 1 VO ίΓΪ N. -ΝΟ χ ;ν Ν-7 I-9b 54 81 I VO Ρϊ N. Ν Ο X ,Ν N-V 1-19 thirty 82 ί fVO Ρϊ N. Ν Ο X ,Ν N-V 1-19 31 83 F (Α ^ν JL JL JL FX Ν. Ν Ο X _Ζ/ Ν NV I-9b 81 84 Λν\4 hi Ν Ο F ri/ I-9b thirty 85 J^QXXjTX N. .Ν Ο χ ;.ν Ν-ί/ I-9b 22 86 ^ΑτνΦγφ l\L Ν Ο F Ύ/ 1-25 41 87 /GK . ^Χ Ν'^^Ν _Ο /^Χ^° \^Ν I-9b 65

-48039788

- 49 039788

98 N. JM O' x > N-Y I-9b [859851-00-6] 60 99 Η'ίΠ Ν Ν Ο X > N-U I-9b [139022-25-6] 24 100 f Υ> ίί№\ Ν. Ν Ο Χ,, ^Ν NV I-9b [1372924-05-4] 53 101 ί 'Ό ρϊ ArAbA^ N. _Ν Ο χ ;.ν N-Y I-9b [1011264-07-5] 28 102 CI ϊ 'П Λ N. Ή0 χ ,} N—U I-9b [1427392-05-9] 61 103 hi Ο nJ^ I-9b [1247451-23-5] 48 104 ϊ Υ^ ¹ N. -Ν Ο χ; ν N-U I-9b [154235-77-5] 21 105 ϊ γ> rQ N. Ν Ο ου I-9b [4790-79-8] 60 106 ϊ ίίΥΛ ρΑ,ζ^Αχ^ΑΑν II Ί Π Η ^Ν - W Ο Χ,Ν NV I-9b [1670-82-2] 76

- 50 039788

107 ΐ ‘Ό ιίι N. 0 Υ/ I-9b [1011264-06-4] 47 108 ^χ ο f Υ> A 1 _____ j< JI >< JL F---TiY^------ N. -N O x; n N-Y I-9b [15855-06-8] 49 109 O / “<7 /°y—z' Vz I-9b [90649-78-8] 21 110 ^f v> A N. _N O x ;n N-Y I-9b [1803602-19-8] 51 111 Cl _f a _t JO _v u M -N O Ύ ;n N-U I-9b [51-44-5] nine 112 F F 'S'---Ί \sSX _p a^^LAJ^ ^{nh F} u'η Π l\LN O x ;n N-if I-9b [23077-43-2] 41 113 14___O^ ⁴ ri/ I-9b [1256820-02-6] 42 114 i Y> irO O ' Ν-α I-9b [16136-58-6] 64

- 51 039788

115 ίΧ N. .Ν Ο Ύ ,Ν N-V I-9b [120512-59-6] 64 116 F Χ/Χ ΖΜ 1^X7XX> N. Ν Ο Χ/Ν Ν-ί/ I-9b [15112-41-1] 73 117 Χ^χ Xu x^xzx^ ^N 'sXXx and 'ί Π Ν. Ν Ο T/ I-12a [120512-59-6] 27 118 χ χχ χχΑ N. Ν Ο I/ I-9b [1248462-73-8] 46 119 ϊ ν> νθ 1 JL Ν AL ^Ε ΊΓί^Γ ' Ίτ ^s Ν. ^4 Ο X > n-ί/ I-9b [3133-78-6] 67 120 .MΧά N. Ν 0 I/ I-9b 45 121 F F^O N. ΉΟ Ύ )Ν N-if I-9b [1427355-37-0] 65 122 F -------j ff^N N. -ΝΟ χ ;ν N-Z< I-9b [1256790-25-6] 36 123 “Ο y° I-9b [6613-44-1] 38

- 52 039788

124 N. _N 0 1 χ ;ν Ν—ν I-9b [470702-35-3] 60 125 ^h o Λ/ύΟυόο Ν. Ο χ ;ν N-# I-9b [15733-83-2] 74 126 F[0 v! .ν ο Ύ/ I-9b [14282-78-1] 35 127 f |< Π ηΓ ^- N. Ο χ > N-# I-9b [13452-14-7] 29 128 4 -π Ο— I-9b [319-60-8] 63 129 ι irV JL ___ JL J! ^<L ^F ^X^r—N. Ν Ο Ύ/ I-9b [1427392-05-9] 44 130 Ι ΊΟ jOf ^{cl F} 0ix^ ^N V^° ^x Ν. _Ν Ο X // ^Ν NV I-9b [85740-98-3] 63 131 luo-L N. Ο X // ^Ν NV I-9b [78621-81-5] 46 132 F LTSS1D N. .Ν Ο χ ;ν N-V I-9b [1211590-99-6] 45

- 53 039788

133 1 ίίίΎ ^ο ϊ JL J. JL -' ^F Ν. Ο X Λ ^Ν Ν—U I-9b [162401-65-2] 61 134 1 F --ΥΥ Ν. Ν Ο X // ^Ν Ν—U I-9b [884494-85-3] 59 135 -ν№% I-9b 53 136 N. L Ο one/ I-9b [56311-39-8] 53 137 ο X ,γ N—V I-9b 4, 6- Dimethylpyridine - 2-carboxylic acid [18088-10-3] 34 138 f< L 0 χ> Ν-7 1-19 Benzofuran-5carboxylic acid [90721-27-0] 25 (combination 63%, 40% SFC) 139 ΛΥΨ" N. .N. 0 χ> N—U 1-9b 1-Methyl-1Hpyrazole-3carboxylic acid [25016-20-0] 67

-54039788

140 And JL0 1-25 3- Methylbenzofuran 2-carbonyl chloride [2256-86-2] 36 141 ML Ύ> ⁰ N-£f I-9b 2, 6- Dimethylpyrimidine -4-carboxylic acid [54198-74-2] fourteen 142 •MIND" Ν..Ν about Kommersant 1-9b 3-Aminopyridine4-carboxylic acid [7529-20-6] 59 143 0 1 . HCl 1-9b 2, 6- Dimethylnicotinic acid [5860-71-9] 44 144 Λίν^/Χν vs ° Ν-ί< 1-9b 5-(tert-Butyl)isoxazole-3-carboxylic acid [90607-21-9] 52 145 v! ή o 1 Ύ/ 1-9b 4-Methyl-2Н-1,3benzodioxole-5carboxylic acid [162506-58-3] 60

- 55 039788

146 F?? N.JN Oh Ύ/ I-9b 4Hydroxyquinoline3-carboxylic acid [34785-11-0] nineteen 147 AkDgk n! h o ci I-9b 3-Chloro-4methylthiophene-2carboxylic acid [229342-86-3] 52 148 kk^k-k' N. N OFF one/ 1-9b 2-Fluoro-3(trifluoromethyl)benzoic acid [115029-22-6] 63 149 n! n o f B > N-^ 1-9b 2, 6Difluorobenzoic acid [385-00-2] 54 150 dkDD N. N 0 NH ₂ 1/ 1-9b 2- Aminonicotypic acid [5345-47-1] 67 151 LCD N. J* 0 ¹ Ύ/ 1-9b 2-Methyl-1,6naphthyridine-3carboxylic acid [387350-63-2] 75

- 56 039788

152 I-9b 3- Methylisoxazole- 5-carboxylic acid [4857-42-5] 63 153 I-9b 5-Bromopyridine-2carboxylic acid [30766-11-1] 45 154 .Cl ιYOYY Yrr 1/ 1-9b 5-Chloropyridine-2-carboxylic acid [36070-80-1] 65 155 ι ίΓ^ΐ XrvXrV N. .Ν 0 F X > N-^y 1-9b 3-Fluoro-4pyridinecarboxylic acid [131307-35-2] 60 156 _r A^O<y' N. Ν 0 ¹ Ύ> Ν—U 1-9b 4-Methyloxazole5-carboxylic acid [2510-32-9] 65 157 N. .N 0 X > N-V 1-9b 5- Methylisoxazole3-carboxylic acid [3405-77-4] 65

- 57 039788

158 nh ₂ hDOD Ν. Λ 0 Ν-^ I-9b 2-Aminothiazole-4carboxylic acid [112539-08-9] 68 159 aDDDD Ύ/ I-9b 2-Chloro-3methylisonicotinic acid [133928-73-1] 54 160 DBP 1-9b 4- Methylisoxazole- 5-carboxylic acid [261350-46-3] 52 161 F'Ύ 1 1D JM Ο F—V X/' 1-9b 2-Methyl-5(trifluoromethyl)oxazole-4carboxylic acid [18955-88-9] 68 162 4> ⁰ N—V 1-9b 6-Aminopyridine2-carboxylic acid [23628-31-1] 53 163 N .vA N. N0 x/ 1-9b 4-cyanopyridine2-carboxylic acid [640296-19-1] 67

- 58 039788

164 N. ⁰ b I-9b 1-Methyl-1nimidazole-5carboxylic acid [41806-40-0] 69 165 AbJ NL -N 0 0. Ύ/ I-9b 5-tert-Butyl-2methoxybenzoic acid [73469-54-2] 61 166 AjOySJ hi n o V ^N 1-9b 2H,3NPyrazolo[3,2B] [1,3]oxazole 6-carboxylic acid [1239722-75-8] 62 167 •AAA® b<>1 0 Kommersant 1-9b 6- (Trifluoromethyl)nor kotic acid [231291-22-8] 48 168 I Y>rr ^OH N. N O 1-9b [350-29-8] 25 169 nL ή o f nJ 1-9b [1737-36-6] 67 170 _% _____ Ck. N. N O 1/ 1-9b [99058-34-1] 69

- 59 039788

171 Oh woA N.N Oh N—U I-9b [946-13-4] 40 172 \ O ϊ rO 1 JL n J4 ^F WY ' A Ύ,Ν N-Y I-9b [59908-54-2] 58 173 .N.___,O H ACOuCC X Xv o x ;n N-U I-9b [54127-63-8] 31 174 F --XX i I'M ^F Η 'ί Π χ N O b I-9b [180283-66-3] 64 175 χ υύ ha hL-N O' N-/ I-9b [872091-00-4] 59 176 ί Ά ^F AiY^ ^N YrjN χ _N O O-U x ;.n N—U I-9b [208772-24-1] 59 177 / F -------S ^-N ^F η Ύ n χ -N O Ύ ;n N-U I-9b [5952-92-1] 75 178 ϊ 'ϊ 1 ίΑ ^F A^r^ ^N ΎΎγ χ JSI 0 №/ Ύ/ I-9b [1187732-69-9] 66 179 ρΛτΧθΧΧ χ -N Ο X-Z Ύ,Ν N-U I-9b [55365-04-3] 68

- 60 039788

180 Cl ^F Υ** ^{4 s} -^ nL _n o Y/ I-9b [24065-33-6] 66 181 F 4/\ NH ₂ N. hl Oh I-9b [3167-49-5] 63 182 Forest® N.h| O one/ I-9b [54045-76-0] 65 183 Ϊ P P ^F Ίι^Γ^ PT? N. hl O N \u003d / x _z \u003e N-U I-9b [208772-23-0] 48 184 UM N. J\l 0 ' b ^m I-9b [17153-20-7] 67 185 F Y> V\ , YU/F ¹ N. N O 1/ I-9b [126909-38-4] 56 186 LAPDH N. hl Oh χ ;.ν N-U I-9b [51446-31-2] 24 187 ^F ΥΧ ^Ν '°\ ΑρΑυΑ) Ν. .Ν Ο ^==/ χ ;.ν Ν—Y I-9b [28691-47-6] 58 188 aAS%XX?- N. Ο X no. n-dj I-9b [90322-32-0] 58 189 v I-9b [1017778-60-7] 26

- 61 039788

190 F ^r Η 1 If =$N Ν. Λ О Ύ > NV I-9b [327056-74-6] 62 191 N. .Ν Ο F I-9b 60 192 ^F y ^F hi. _ν o Ύ/ I-9b [886496-49-7] 21 193 LHOiSSH N. J4 O bp I-9b [1214383-15-9] 57 194 f XXX kwCMX< Μ Ν Ο F M< ^N I-9b [67515-55-3] 25 195 F XXX ZX^ ^F A/COjCX N. Ν O x,> N-U I-9b [82846-18-2] 76 196 1 f 'Mb /M i ^Γ i ii ^xr I-9b [26218-80-4] 45 197 N _P A6X+'>A^ N. Ν O Ύ ,N NV I-9b [1877-72-1] 22 198 F --------> ^N ^Z pA^JQXQ ^{n F} Η π Π \ Ν. N 0 ' Y/ .HCl I-9b [113100-61-1] 49

- 62 039788

199 ^N \^ ^N 1-42 8.7 200 F V2 ₀ / *s 1 Vk LN—\ \—F 1-30 54 201 F 4¼ 1-30 thirty 202 vco 1-42 31 203 F \—F ^N= \ 1 N—/ ^N 'Si o' I-30a 87 204 F \--F N^=( f N—X y--^m ^z Vs \ O I-30b 89

- 63 039788

205 1-12 22 206 1-3b 60 207 ddd. \X*s 1-3b 40 208 Ν O C %--NF TDD 'X/ ν-\=/ ' ^F \ X ^s 1-3b 75 209 l.o 1 Lie F 1-3b 57 210 F 1°Xik. X 1 D ^N k?r XY O'- 1-3b 65 211 DD 1-3b 25

- 64 039788

212 ^F \ 7 1 °v\ 7 < ^N N^N^7*S 7 \=N * 1-12 fourteen 213 Ν π — ^{Ν F} W \\^ )=/ F θ 7----< \\ / ^R \ y — Ν ^S / — -Λ ^S I-9a 61 214 Ν ίΐ - ^NF \W )=/ F °w A\ V—N s\— I-9a 55 215 n .K ₌ 'n^n JL .F L Y f coL ⁰ F I-9a 54 216 ^N >=N £>a VnH \=/ -W Y \ 7 - O '----' I-9a 29

-65 039788

- 66 039788

221 X Μ / ^ω \__/ \ \ _ζ _/ν/ °^\ / / ⁴¹ / ° ⁴¹ I-9a 58 222 Ν μ —ν f NN /=^ F Ο , k \—Ν r\ Ν=/ '' Xu ^F I-9b 24 223 Ν ί| 'Ί--- ^Ν F ^ν -ν ν_ζ /=/ F ο ,—4 χ—Ν r\ Μ FX .1.30 HC1 . Η ₂ Ο I-9b 37 224 N. 0F Ύ ;ν n~U I-12a 55 225 a'OCM? N. JM OF Ύ ;n N-U I-12a 58

- 67 039788

226 N (| %—N F W \\^ th ' \—N \=O I-9b fourteen 227 N. ⁴ N^^N 0 ^iM r jCj ° \=N I-9b 49 228 /Tu ^N Jv , ⁴ N ^/ ^'N Qvy Gd ⁰ Vj I-9b fifty 229 N ίι — ^NF n—n \V^ ° y—4 y—N r\ O I-9b 83 230 ^h'r 1-25 32

- 68 039788

231 n d ^,v v jAj ° ΗΟ®γ II N I-9b 29 232 N 0 J-- ^NF N-/ -ζ /=/ F O y-Zs y-N ) / \ / ^R iJ \l I-9b 65 233 N J--NF N^/ -ζ A \--N y=° N--/ C ,/ ^nh2 I-9b 57 234 N ii A— ^NF nJ/ \\^ J '--N \=° Cy I-9b 63

-69039788

235 Ν Π -- ^N F N-/ --ζ b ''---N \=O {U ^C| N— I-9b 62 236 N 0 -- ^NF N-/ b-( 8'''---N \=° Ζ N D HO I-9b 2 237 Nf| b- ^NF c/ \\D u ''-Ν \=Ο Ν=Ζ 4 w ν-Ν I-9b 54 238 Μ 0 -- ^{Ν F} '---Ν /=° ^Ν =\ I-9b 49

- 70 039788

239 Ν C --NF Υ/ \W Ϋ ' \--Ν 2=° Ν---' _F I-9b 45 240 .Ν ίι 5— ^{Ν F} NY Η '>—Ν \=0 Ν=/ I-9b 59 241 “Π Ο I-9b 66 242 ζΎ>Ί ο ο I-9b 67

- 71 039788

243 Ν ίι 2 - ^NF \=° A o I-9b 44 244 N (1 2 - ^NF N-/ Y-( )=/ F o /--V Y-N r\ ^ ⁼ⁿ I-9b 53 245 / G8 ^N 32. ⁴ N^^N ό·^' £2° F I-9b 62 246 N f| —N F n-V /=/F ---v 0 y--- \—0 y—N R>...... tr I-9b 58 247 ^N 32. tW F O =4° I-9b 53

- 72 039788

248 N Jk ⁴ N' ^/X 'N F GD ⁰ N D rr I-9b 63 249 /P N Jk c%y N^ky^^O [1 J I-9b 53 250 N 0 -- ^N F ^N -NW % ''---N / ^=OF <J ^N I-9b 66 251 /Tu ^N >k , ⁴ N^^N Dr^ ⁰ I-9b 70

- 73 039788

- 74 039788

257 ν _όΛν Μ I-9b 59 258 Μϊ frou ~4~€/ι ° 1 Ο- ^Ν I-9b 53 259 >ΤΚ Ν Jy ό^· / MqAc I-9b 63 260 Ν ίι — ^{Ν F} Ν-V \\^ /=/ ^F ° y--- \)^ ^Ν νν ^κ Ν I-9b 40 261 /Τϊ ^Ν 3κ , ^Χ Ν' ^Χ ^Ν ό^' 0 ^x ^bj Xn ₂ I-9b 64

- 75 039788

262 /O ^N Jk όΑΥ γΟ° ^N-^^F I-9b 68 263 Ν ίι — ^{Ν F} W \\^ /=/ F ο ,—φ \J0 ⁿ vJ4 /A ^F \/ zu F I-9b 27 264 Ν (ι - ^{Ν F} Ν-/ \\^/ /=/ F O >----(s xO I-9b 58 265 ^N η 9— ^NF NV /=/ ^{F 0} /—V y—N >...... / \ /R ⁿ ^O O \ I-9b 62

- 76 039788

266 N to ^Ν HELL] ° Ν. BUT I-9b 72 267 Ν Ν 0 X? I-9b 57 268 ^Ν A 0 ^:V V Cl Ι\Γ Ν AL ⁴ α I-9b 61 269 ν ίι — ^{Ν F} W Α-ζ \ /=/ ^F )-----i's / ^R \ '---Ν /=° AID- ^F Ν—J I-9b 61 270 /\ΟF qA' N=/ Cl I-9b 45

- 77 039788

- 78 039788

277 ^p Ar%PMA N. _Ν Ο F Χ,Ν N-U I-19a 71 278 hL -Ν 0 F ^ν ύ ;ν Ν—V I-9b fourteen 279 υαΑ N. .Ν Ο F ύ Ν—V I-9b eleven 280 αυΑα N. .Ν 0 0. ^ν χ _ζ > Ν—V I-9b 3 281 f fT^ ^NF JL JL χ JL AL JL ^F ^ll^l^ ^X - ^F l\LN O y ;.n NV I-9b thirty 282 F ^AA rf^N F ^x A^l ⁴ ”^ Yl ° l\L J4 O F “ // ^N NV I-9b 24 283 ^N X> ° f^f NV I-12a 48

- 79 039788

- 80 039788

290 fYvCy ¹ ^ |\LN 0 1-2 5a 40. 291 n! X ⁰ N—V I-25a 65 292 hL N 0 b ^m 1-2 5a 59 293 N. .N0 N-V 1-2 5a 65 294 |\L .N 0 F X > N—'J 1-2 5a 60 295 hL .Ν O Ύ/ 1-2 5a 94

-81 039788

296 Ϋ° 1-2 5a 39 297 N. N o X ,/ N-U I-25a 61 298 N. J4 0 ¹ Ύ/ 1-2 5a 56 299 N. .N 0 Ύ> N-U 1-2 5a 54 300 ν' A ⁰ N—V 1-2 5a 22 301 O ^X fAY^Y^ ⁰ '' N. .Ν O X/ 1-2 5a 60

- 82 039788

302 KL JM 0 b ^m 1-2 5a 61 303 yes/d' N. .N Oh N-U 1-2 5a 41 304 λΥ ν' A 0 N-U 1-2 5a 56 305 ν' h 0 o-U Ύ> \ N-U 1-2 5a 64 306 N.J+° Ύ/ 1-2 5a 68 307 aW NL-N O Ύ> N—U I-25a 55

- 83 039788

308 M N 0 N-U 1-2 5a 48 309 N. .N 0 N-U 1-2 5a 65 310 CI _Ρ Α _ΊΊ ^ζψΟ0 Ν. .N 0 N—V 1-2 5a 52 311 va-co 14 N 0 N-V 1-2 5a 62 312 VA-OA Ύ>” 1-2 5a 63 313 l\L N 0 N-U 1-2 5a 63 314 X F M. .Ν Ο Υ), ^Ν Ν—U 1-2 5a 64

- 84 039788

315 N-U 1-2 5a 63 316 N-U 1-2 5a 100 317 AqAV6 N-U 1-2 5a 61 318 Ύ> N-U 1-2 5a 86 319 ν' A 0 Ύ> Ν-ΖΖ 1-2 5a 57 320 ΑοΑνΑ Ύ> Ν—U 1-2 5a fifty

- 85 039788

321 2=0 y-z \sch / )—χ z. /ω\/ 7-Z I-9b (Note: Combination via EDCI) 60 322 ΜΙ h F 1 ίΐ Ί 1 si i 1J N. N 0 ύ> N-V I-9b (Note: Combination via EDCI) 69 323 F Ύ> νΧζ 1 si ! JU D N. .Ν ⁰ Ύ> 1-9b (Note: Combination via EDCI) 59 324 ΐ sPI ί|Ί 1 SI _Ν II Ν π Ν. .Ν 0 Ύ>Ν-Ζ< I-9b (Note: Combination via EDCI) 79 325 χί L z—h « 1^. / \ ω ? ^ζ /“Κ / ^π \ ζ—' °Ж I-9b (Note: Combination via EDCI) 62

- 86 039788

326 F p > ί| ίΓ V NL .N 0 Ύ> Ν—V I-9b (Note: Combination via EDCI) 66 327 N.N.O Ύ/ I-9b (Note: Combination via EDCI) 73 328 : O 1 si A jLn— ^F i/ 1-9b (Note: Combination via EDCI) 56 329 o J=o Y--z. 4a V- z ^z I-9b (Note: Combination via EDCI) 71 331 /0 /K f η <= X Ji J Π l\L .N 0 Ύ> N—U I-9b (Note: Combination via EDCI) 66

- 87 039788

332 F mi _H f Yr η if Μ X Ji J NL Ν O dq Ν—V I-9b 32 333 Cl f/yv^V^ ⁰¹ N. Ν O Ύ/ 1-2 5a (Note: phosphonic anhydride acids) 61 334 Cl dC&D N. Ν O Y /G Ν—V I-9b (Note: phosphonic acid anhydride) 66 335 ___yA^N LchF-M N.Xv° x/ 1-2 5a 71 336 luCmx hL N 0 Y// ^N Ν—V 1-2 5a (Note: phosphonic anhydride) 52 337 _F ^vCM^ Ύ> ° 1-2 5a 62

- 88 039788

338 AY |\L -Ν ⁰ Ύ _/Ζ ^Ν Ν—U 1-2 5a 33 339 Κ Ν 0 1-2 5a 69 340 W4 ^, N. Ν 0 Ύ ν Ν—U 1-2 5a 38 341 F N. N 0 X No. Ν—U 1-2 5a 72 342 F F^^F K N 0 X if Ν—U 1-2 5a 42 343 K AL 0 x> N-Y 1-2 5a (Note: phosphonic anhydride acids) 58

- 89 039788

344 N. .N 0 1-2 5a 64 345 ⁰ 1-2 5a (Note: phosphonic acid anhydride) 42 346 N. .N. 0 Ύ/ 1-2 5a 41 347 N. N 0 Ύ> Ν—U 1-25a 52 348 pY^SM^ N. N 0 one/ 1-2 5a 51 349 Ν JM 0 Ύ/ 1-25 46 350 N. N 0 v > N-U 1-2 5a 55

- 90 039788

351 N. .N 0 1-2 5a 352 F F'^O N. N 0 ^v N—V 1-2 5a 67 353 N. .N 0 Ύ> Ν—V 1-2 5a 66 354 dDO-D ^- NN 0 Ύ 3 N—V 1-2 5a 59 355 N. .N 0 Ύ> N—U 1-2 5a (Note: phosphonic anhydride acids) 34 356 LAD N. L 0 one/ 1-2 5a 78 357 A^CvCQ ν' X 0 F b ^s 1-2 5a 65

- 91 039788

358 N. .N 0' 1-2 5a 17 359 AaCv^ ^f N. M O 1-2 5a 58 360 ^s x N. .N O Ύ I N—U 1-2 5a 43 361 VAX hL N O I-9b fifteen 362 XaCvY l\L N 0 Ύ> N-U 1-2 5a 47 363 KL Al 0 X > N—Y 1-2 5a 49

- 92 039788

364 N. N ⁰ 1-2 5a 68 365 KL Ν 0 b ^m 1-2 5a thirteen 366 KL Ν 0 b ^m 1-2 5a 59 367 N. ΚΙ Oh one/ 1-2 5a 68 368 s-^ hL N O Vff N-U 1-2 5a fifty 369 Cl l\L N 0 V ,γ N-U 1-2 5a 26

- 93 039788

370 K N 0 Ύ/ 1-2 5a 60 371 N. N 0 Ύ> N-V 1-2 5a 34 372 Ατν^M^ N. N 0 N-U I-25a 37 373 K Ν Ο X ρ N-V 1-2 5a 63 374 ρΑϊ^Φ'ι/Χ N. .Ν Ο Ύ> N-V 1-2 5a 53 375 N. .N 0 Ύ> Ν—ν 1-2 5a 71 376 Κ Ν 0 Ύ> Ν—ν I-25a 57

- 94 039788

377 Κ X O ^m 1-25 41 378 M JM 0 b ^m 1-25 58 379 / Η Ί s P N. L O N-U 1-2 5a 63 380 DBP K N O N-U 1-2 5a 68 381 K N O Ύ>N—V 1-2 5a 59 382 l\L L O one/ 1-2 5a 54 383 ν' A O Kommersant 1-25a 55

- 95 039788

384 -j\ch.r—ζ' /1 \ω ζ ω yr 1-2 5a 22 385 N. Ν O N-U 1-2 5a 72 403 / N__ f (7^Ί ³ ίι χΓ ^fZ I|Ns nJr Ν. Ν О F Ύ if N—U 1-2 5a 57.7 404 N. Λ° Ύ if n-Lf 1-2 5a 67 405 .L ^N -- F ίΐΎ ^F li^is N—U 1-25 68.7 406 ⁰ 1-25 71.5

Conversions and synthesis of final compounds by other routes Compound 386 and Compound 282/387.

Comm. No. 386 Conn. No. 387/282

Step 1 Compound 388 and Compound 389

CI

Comm. No. 388

To a solution of compound 71 (654 mg, 1.42 mmol) in MeOH (13 ml) was added NaOMe (10.5 ml, 56.7 mmol, 30% in MeOH) and the resulting mixture was stirred at 0°C for 1 h. reaction see quenched with saturated aqueous NH ₄ C1. The volatiles were removed under vacuum and the remaining aqueous residue was separated using DCM. The aqueous layer was extracted with DCM (2x10 ml). The combined organic layers were dried over MgSO ₄ , filtered and evaporated in vacuo to give a mixture of compounds-96039788 388 and 389 as a white foam (656 mg) which was used as such in the next step.

Step 2 Compound 386 and Compound 282/387.

The mixture from the previous step was dissolved in 1,4-dioxane (10 ml), the resulting solution was placed in a microwave reaction tube and degassed for 5 minutes. K ₂ CO ₃ (668 mg, 4.83 mmol) was then added followed by trimethylboroxine (0.789 ml, 2.76 mmol, 3.5 M in THF) and Pd(PPh ₃ ) ₄ (159.5 mg, 0. 14 mmol). The reaction mixture was then heated at 100° C. for 2.5 h under microwave radiation. Additional trimethylboroxine (0.394 ml, 1.38 mmol, 3.5 M in THF) was added and the reaction mixture was heated at 100° C. under microwave radiation for an additional hour. The solvent was evaporated in vacuo and the resulting residue was dissolved in a mixture of DCM/water 1:1 (40 ml). The biphasic mixture was separated and the aqueous phase was extracted with DCM (2x10 ml). The combined organic layers were dried over MgSO ₄ , filtered and concentrated under reduced pressure. The resulting residue (843 mg) was purified by silica gel flash column chromatography using a DCM/MeOH gradient from 100/0 to 97/3 as eluent. Fractions corresponding to the title compounds were collected. One fraction was recrystallized from DIPE to give compound 282/387 (146 mg, 24%). The second fraction was purified by preparative HPLC using as stationary phase: RP XBridge Prep C18 OBD-10 µm, 30x150 mm, and as mobile phase: 0.25% solution of NH4HCO3 in water, CH3CN to give compound 386 as a white solid substances (82 mg, 13.8%).

Connection 390 and connection 391.

Comm. No. 390 Conn. No. 391

Compound 271 (150 mg, 0.37 mmol), cyclopropylboronic acid (127.2 mg, 1.48 mmol) and toluene (3.0 mL) were charged to a pressure reaction tube and degassed for 15 min before successive addition of palladium(II) acetate (4.1 mg, 0.0185 mmol), tricyclohexylphosphine (10.4 mg, 0.037 mmol), distilled water (0.734 ml, 40.65 mmol) and tribasic potassium phosphate (235.7 mg, 1.11 mmol). The tube was stoppered, reaction. cm. was placed in an oil bath with a temperature of 100°C and stirred for 16 hours. The resulting solution was cooled to rt. and filtered through a pad of Celite®, which was washed with toluene and EtOAc. The filtrate was evaporated to dryness under reduced pressure. The resulting residue was dissolved in EtOAc and washed with water and brine, then dried over MgSO ₄ , filtered and evaporated in vacuo. The resulting residue (155 mg) was purified by preparative HPLC using as stationary phase: RP XBridge Prep C18 OBD-10 µm, 30x150 mm and as mobile phase: 0.5% NH4Ac in water+10% CH3CN, MeOH , to give compound 390 (18 mg, 11.7%) and compound 391 (bb mg, 43.4%) after trituration with heptane.

Compound 392.

Stage 1. Compound 393.

CI

To a solution of compound 71 (436 mg, 0.95 mmol) in DMF (7.35 ml) was added cesium fluoride (577 mg, 3.8 mmol) and the resulting mixture was stirred and degassed for 15 min. Cyclopropanol (0.072 ml, 1.14 mmol) was added and react. see heated at 110°C for 2 h under the action of microwave radiation. The solvent was removed in vacuo, then the resulting residue was dissolved in a mixture of DCM/water 1:1

- 97 039788 (20 ml). The resulting biphasic mixture was separated, then the aqueous layer was extracted with DCM (2x10 ml). The combined organic layers were dried over MgSO ₄ , filtered and the solvent was removed under reduced pressure. The resulting residue was purified by silica gel flash column chromatography using a DCM/MeOH gradient from 100/0 to 99/1 as eluent to give compound 393 (308 mg) as an impure substance, which was used as such in the next step.

Stage 2. Compound 392.

A solution of compound 393 (308 mg, 0.64 mmol) in 1,4-dioxane (4.6 ml) was degassed for 15 min, then K ₂ CO ₃ (310 mg, 2.24 mmol), trimethylboroxine ( 0.550 ml, 1.92 mmol, 3.5 M in THF) and Pd(PPh ₃ ) ₄ (74.015 mg, 0.064 mmol) and the resulting mixture was heated at 100°C for 2.5 h in a pressure test tube . The solvent was removed in vacuo and the resulting residue was partitioned between DCM (10 ml) and water (10 ml). The resulting biphasic mixture was separated and the aqueous layer was extracted with DCM (3x10 ml). The combined organic layers were dried over MgSO ₄ , filtered and the solvent was removed under reduced pressure. The resulting residue (340 mg) was purified by preparative HPLC using as stationary phase: RP Vydac® Denali® C18-10 μm, 200 g, ID 5 cm, and as mobile phase: MeOH, to give compound 392 (55 mg , 18.6%) as white crystals after recrystallization from DIPE.

Connection 394.

Stage 1. Compound 395.

To a stirred suspension of intermediate 9-b (750 mg, 2.47 mmol) in DCM (20 mL) were added successively EDCI (993 mg, 5.2 mmol) and 6-chloro-3-fluoro-2-methylisonicotinic acid (intermediate compound 50, 492 mg, 2.6 mmol). DIPEA (1.78 mL, 10.4 mmol) was then added to the mixture and the resulting yellow solution was stirred at rt. within 5 hours Reaction. see quenched with aqueous NaOH solution (20 ml, 1 M). The aqueous layer was extracted with DCM (3x10 ml) and the combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The resulting residue (1.5 g) was purified by silica gel flash column chromatography using a DCM/MeOH gradient from 100/0 to 99/1 as eluent to give compound 395 (720 mg, 62%) as a white foam.

Stage 2 Intermediate 56.

Iodotrimethylsilane (0.065 ml, 0.46 mmol) was added to a solution of compound 395 (200 mg, 0.46 mmol) in propionitrile (0.8 ml) under nitrogen atmosphere. Then added sodium iodide (205 mg, 1.37 mmol) and the resulting solution was heated to 80°C. After 5 hours, LCMS analysis showed only 15% conversion, so another portion of iodotrimethylsilane (0.065 mL, 0.456 mmol) was added and react. cm was stirred at 80°C for 22 hours. After this time, another portion of iodotrimethylsilane (0.065 ml, 0.456 mmol) and sodium iodide (205 mg, 1.37 mmol) was added and the reaction. see was stirred for 12 hours Removed the solvent in vacuo, then the resulting residue was divided between water and DCM. The resulting biphasic mixture was separated and the aqueous layer was extracted with DCM (3x20 ml). The combined organic layers were dried over MgSO ₄ , filtered and the solvent was removed in vacuo. The resulting residue (1.5 g) was purified by silica gel flash column chromatography.

- 98 039788 using a DCM/MeOH gradient from 100/0 to 97.5/2.5 as eluent to give Intermediate 56 (162 mg) as an impure substance, used as such in the next step.

Stage 3. Connection 394.

Methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (0.192 ml, 1.51 mmol) was added to a mixture of intermediate 56 (160 mg, 0.3 mmol) and copper iodide (75 mg, 0.39 mmol) in anhydrous DMF (0.958 ml) in a pressure reaction tube. The resulting mixture was heated to 80° C. for 2 hours in an oil bath. reaction see cooled to rt, then extinguished with a saturated aqueous solution of NH ₄ Cl. The resulting mixture was extracted with EtOAc (3x15 ml). The combined organic layers were dried over MgSO ₄ , filtered and the solvent was removed in vacuo. The resulting residue was purified by flash column chromatography using a 100/0 to 60/40 heptane/EtOAc gradient as eluent to give compound 394 (17.5 mg, 12%).

Compound 396.

Stage 1. Connection 397.

To a stirred suspension of intermediate 9-b (740.1 mg, 2.44 mmol) in DCM (18.5 ml) was added EDCI (934 mg, 4.87 mmol) and 2-chloro-3-fluoro-6-methylisonicotinic acid (485 mg, 2.56 mmol). DIPEA (1.68 ml, 9.74 mmol) was added and react. cm. stirred for 2 h at room temperature. reaction see quenched with aqueous NaOH solution (30 ml, 1 M). The aqueous layer was extracted with DCM (3x15 ml) and the combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The resulting residue (1.5 g) was purified by silica gel flash column chromatography using a DCM/MeOH gradient from 100/0 to 99/1 as eluent to give compound 397 (854 mg, 80%) as a white powder.

Stage 2 Intermediate 57.

Iodotrimethylsilane (0.26 ml, 1.82 mmol) was added to a solution of compound 397 (400 mg, 0.91 mmol) in propionitrile (1.6 ml) under nitrogen atmosphere. Sodium iodide (410 mg, 2.73 mmol) was then added and the resulting solution was heated to 80° C. for 1.5 h. The solvent was removed in vacuo, then the resulting residue was partitioned between water and EtOAc. The resulting biphasic mixture was separated and the aqueous layer was extracted with EtOAc (3x20 ml). The combined organic layers were dried over MgSO ₄ , filtered and the solvent was removed under reduced pressure. The resulting residue was purified by silica gel flash column chromatography using a DCM/MeOH gradient from 100/0 to 97.5/2.5 as eluent to give Intermediate 57 (197 mg) as an impure substance which was used as such in the next stage.

Stage 3. Connection 396.

To a solution of intermediate 57 (197 mg, 0.37 mmol) in anhydrous DMF (1.18 ml) was added copper iodide (92 mg, 0.48 mmol) and the resulting mixture was degassed with nitrogen for 10 minutes. Then methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (0.236 ml, 1.86 mmol) was added and react. cm. was stirred and heated at 80°C for 2 hours, after which it was left to cool to rt. during the night. reaction see quenched with saturated aqueous NH ₄ Cl. The resulting mixture was extracted with EtOAc (6x20 ml). The combined organic layers were dried over MgSO ₄ , filtered and the solvent was removed in vacuo. The resulting residue (550 mg) was purified by preparative HPLC using as stationary phase: RP XBridge Prep C18 OBD-10 µm, 50x150 mm, and as mobile phase: CH3CN, to give compound 396 (7.4 mg, 4%).

Compound 398.

Stage 1. Connection 399.

To a stirred solution of intermediate 9-b (242 mg, 0.8 mmol) and 2-chloro-3-fluoro-6methoxyisonicotinic acid (intermediate 53, 172 mg, 0.84 mmol) in DCM (6.35 ml) was added EDCI (320.5 mg, 1.67 mmol) followed by DIPEA (0.57 ml, 3.34 mmol). reaction see stirred at room temperature during the night. Added another portion of EDCI (80 mg, 0.417 mmol) and DIPEA (0.1 ml, 0.75 g/ml, 0.58 mmol) and react. see was stirred for 12 h, then it was extinguished with an aqueous solution of NaOH (20 ml, 1 M). The aqueous layer was extracted with DCM (3x20 ml) and the combined organic layers were dried over MgSO ₄ , filtered and concentrated in vacuo. The resulting residue (600 mg) was purified by silica gel flash column chromatography using a DCM/MeOH gradient from 100/0 to 98.5/1.5 as eluent to give compound 399 (136 mg, 80%) as a white foam.

Stage 2. Connection 398.

A solution of compound 399 (136 mg, 0.299 mmol) in 1,4-dioxane (2.17 mL) was degassed for 15 min in a pressure reaction tube. K ₂ CO ₃ (144.6 mg, 1.05 mmol), trimethylboroxine (0.256 ml, 0.897 mmol, 3.5 M in THF) and Pd(PPh ₃ ) ₄ (34.5 mg, 0.03 mmol) were added successively. ) and the resulting mixture was heated at 100° C. for 2 hours. The solvent was removed in vacuo and the resulting residue was partitioned between DCM and water. The resulting biphasic mixture was separated and the aqueous layer was extracted with DCM (3x15 ml). The combined organic layers were dried over MgSO ₄ , filtered and the solvent was removed under reduced pressure. The resulting residue (220 mg) was purified by silica gel flash column chromatography using a DCM/MeOH gradient from 100/0 to 96/4 as eluent to give compound 398 (88 mg, 68%).

Connection 400.

Stage 1. Connection 401.

DIPEA (0.65 ml, 3.77 mmol) and HBTU (300 mg, 0.79 mmol). Intermediate 25-a (238 mg, 0.75 mmol) was then added and react. cm. stirred for ~1.5 h at rt. reaction quenched with aqueous NaOH solution (1 ml, 1 M) and then filtered through a filter

- 100 039788

Extremelute®. The solvent was removed under reduced pressure. The resulting residue was purified by silica gel flash column chromatography using a gradient eluent

DCM/MeOH 100/0 to 98/2 to give compound 401 (320.5 mg, 83%) as a colorless oil.

Stage 2. Connection 400.

A solution of compound 401 (320 mg, 0.71 mmol) in 1,4-dioxane (5.1 ml) was degassed for 15 minutes. K ₂ CO ₃ (343.3 mg, 2.45 mmol), trimethylboroxine (0.608 ml, 2.129 mmol, 3.5 M in THF) and Pd(PPh ₃ ) ₄ (82.0 mg, 0.071 mmol) were added successively and the resulting mixture was heated at 100° C. for 2 hours in a pressure reaction tube. The solvent was removed in vacuo and the resulting residue was partitioned between DCM (20 ml) and water (20 ml). The biphasic mixture was separated and the aqueous layer was extracted with DCM (3x15 ml). The combined organic layers were dried over MgSO ₄ , filtered and the solvent was removed under reduced pressure. The resulting residue was purified by silica gel flash column chromatography using a DCM/MeOH gradient from 100/0 to 95/5 as eluent. This gave a mixture of endo/exo isomers (230 mg) which was separated by preparative HPLC using as stationary phase: RP XBridge® Prep C18 OBD-10 µm, 50x150 mm and as mobile phase: MeOH. Fractions containing product were evaporated and the resulting residue was recrystallized from Et2O to give compound 400 (57 mg, 18.6%).

Connection 402.

To a solution of compound 1 from WO 2017/076900 (250 mg, 0.624 mmol) in DCM (1.6 ml) was added mCPBA (307.825 mg, 1.25 mmol) at 10°C and the resulting mixture was stirred in Easymax® for 3 days . After this time, the reaction see extinguished with saturated aqueous NaHCO ₃ (1.5 ml). The biphasic mixture was separated and the aqueous layer was extracted with DCM (3x5 ml). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The resulting residue was purified by silica gel flash column chromatography using a DCM/MeOH gradient from 100/0 to 96/4 as eluent to give compound 402 (250 mg, 96%) as a white powder.

Analytical part

Melting point values.

The values are either maximum values or melting point ranges and are subject to experimental uncertainties that are typically associated with a given analytical method.

The melting point was determined using a DSC823e (Mettler-Toledo). The melting point was measured at a temperature gradient of 10°C/min. The maximum temperature was 300°C.

- 101 039788

table 2

conn. no. T. pl. (°C) 22 217.82 23 169.13 25a 240.33 25b 239.55 29 176, 52 31 190.16 40 181.11 49 263.81 52 228.39 69 209.73 72 177.60 77 177.37 79 227.10 93 201.12 102 215.44 106 273.02 108 151.39 111 188.75 116 192.09 118 179.82 120 173.99 122 160.20 123 203.87 126 114.32 127 139.13 129 140.78 130 193.85 131 224.39 132 116.57 133 152.33 137 229.91 189 152.03 191 162.07 192 110, 16 194 167.52 201 140.14 202 78, 65 203 204.03 204 205.47

LC/MS methods.

High Performance Liquid Chromatography (HPLC) measurements were performed using an LC pump, diode array detector (DAD) or UV detector, and a column as

- 102 039788 described in the respective methods. If necessary, additional detectors were turned on (see Fig.

method table below).

The flow from the column was sent to a mass spectrometer (MS), which was equipped with an atmospheric pressure ionization source. It is within the skill of the art to set configurable parameters (eg, scan range, dwell time, etc.) to produce ions to determine the nominal monoisotopic molecular weight (MW) of the compound. Data collection and processing was carried out using the appropriate software. Compounds were described using their experimental retention times (Rt) and ions. Unless otherwise indicated, in the datasheet the molecular ion indicated is [M+H]+ (protonated molecule) and/or [MH] ^- (deprotonated molecule). In case the compound was not directly ionizable, indicate the type of adduct (ie [M+NH4] ⁺ , [M+HCOO]-, etc.). For molecules with complex isotopic distributions (Br, Cl), the reported value is the value that was obtained for the smallest isotope mass. All results were obtained with experimental errors, which are usually associated with the method used.

Throughout this document, SQD stands for Single Quadrupole Detector, MSD stands for Mass Selective Detector, k.t. stands for room temperature, BH stands for ethylsiloxane/silica bridged hybrid, DAD stands for diode array detector, HSS stands for high strength silica.

Table 3A. LCMS method codes (flow rate expressed in ml/min;

column temperature (T) in °C; analysis time in minutes)

THE CODE WAYS device Speaker mobile phase Gradient Flow rate T columns Analysis time (min) BUT waters: Acquity® UPLC®- DAD/SQD Waters : WEN C18 (1.7um, 2.1*50mm) A: 10 mM CH3COONH4 in 95% H2O+5% CH3CN B:CH3CN 95% A to 5% A in 1.3 min, hold for 0.7 min. 0.8 55 2 AT waters: Acquity® UPLC® - DAD and SQD Waters : WEN C18 (1.7um, 2.1*50mm) A: 25mm CH3COONH4 in 95% H2O+5% CH3CN B:CH3CN 95% A to 5% A in 1.3 min, hold for 0.7 min. 0.8 55 2 With waters: Acquity® UPLC®- DAD and SQD waters: HSS T3 (1.8um, 2.1*100mm) A: 10 mM CH3COONH4 in 95% H2O+5% CH3CN B:CH3CN From 100% A TO 5% A in 2.10 min., up to 0% A in 0.90 min., up to 5% A in 0.5 min. 0.7 55 3.5 D waters: Alliance® -DAD - ZQ and ELSD 2000 Speaker Xterra MS C18 (3.5 µm, 4.6x100 A: 10 mM CH3COONH4 in 95% H2O+5% CH3CN From 100% A to 1% A, 49% B and 50% C in 6.5 minutes, up to 1% sixteen 40 eleven

- 103 039788

Alltech mm) B:CH3CN C:CH3OH D: (40% CH3CN and 40% CH3OH and 20% H2O with 0.25% CH3COOH A and 99% B in 0.5 min., up to 100% D in 1 min. with holding m for 1.0 min. up to 100% A in 0.5 min. and with holding m for 1.5 min. E Waters : Acquity® UPLC® DAD-ELSD and SQD Waters : WEN C18 (1.7um, 2.1*50mm) A: 0.1% UNSD+5% CHZON in H2O B: CH3CN From 95% A to 0% A in 2.50 minutes, to 5% A in 0.5 minutes. 0.7 55 3

Table 3B. LCMS-R analysis data _t means retention time (in minutes), [M+H]+ means mass of protonated compound, method means the method used for analysis (LC)MS

CONN. NO. Rt (min.) [M+N] ⁺ [M+N] - Way 1a 1.33 365 363 With 1b 1.33 365 363 With 2a 1.38 393 391 With 2b 1.38 393 391 With Behind 1.43 365 363 With 3b 1.43 365 363 With 4 0.82 419 417 BUT 5 0.87 390 388 BUT 6 0.8 376 374 BUT

- 104 039788

7 0.94 390 388 A eight 0.87 376 374 A nine 0.96 382 380 A ten 0.99 443 441 A eleven 0.76 389 387 A 12 0.84 425 423 A thirteen 0.7 456 514 [M+CH3COO]- A fourteen 0.66 339 337 A fifteen 0.69 371 369 A sixteen 0.85 370 428 A 17 0.82 405 403 A eighteen 0.72 385 383 A nineteen 0.68 391 389 A 20 0.84 444 442 A 21 0.82 377 375 A 22 0.84 401 459 [M+CH3COO]- A 23 1.57 391 389 C 24a 1.85 445 503 [M+CH3COO]- C 24b 1.86 445 443 C 25a 1.54 441 439 C 25b 1.55 441 439 C 26a 1.46 376 374 C 26b 1.46 376 374 C 27 1.65 421 419 C 28 1.73 439 437 C 29 0.72 413 411 A thirty 0.72 413 411 A 31 0.72 413 411 A 32 0.74 401 399 A 33 1.66 447 445 C 34 1.66 447 445 C

- 105 039788

35 1.59 492 490 With 36 0.85 421 419 BUT 37 1, 68 435 433 With 38 1.36 419 417 With 39 1, 84 475 473 With 40 0.69 412 410 BUT 41 0.85 455 453 BUT 42 sixteen 407 405 With 43 0.9 441 439 BUT 44 0.96 443 441 BUT 45 0.98 443 441 BUT 46 0.98 443 441 BUT 47 1.79 413 411 With 48 1, 03 426 424 BUT 49 0.88 401 399 BUT fifty 1.58 427 425 With 51 0.81 421 419 BUT 52 0.9 455 453 BUT 53 0.82 417 415 BUT 54 0.87 425 423 BUT 55 1.7 425 423 With 56 1, 64 434 432 With 57 1.45 376 374 With 58 1.31 376 374 With 59 eighteen 480 478 With 60 1.74 451 449 With 61 0.74 404 402 BUT 62 1.50 417 415 With 63 1.88 452 450 With 64 eighteen 441 439 With 65 1, 84 406 404 With 66 1.53 403 401 With 67 1.53 375 373 With 68 0.78 405 403 BUT

- 106 039788

69 0.96 412 410 BUT 70 0.81 419 417 BUT 71 0.99 459 457 BUT 72 2.02 446 444 With 73 0.95 443 441 BUT 74 1.97 448 446 With 75 1.97 448 506 [M+CH3COO]- With 76 eleven 434 432 BUT 77 0.97 430 428 BUT 78 0.94 425 423 BUT 79 0.82 432 430 BUT 80 sixteen 415 413 With 81 1.57 419 417 With 82 1.58 419 417 With 83 0.9 421 419 BUT 84 0.78 405 403 BUT 85 1, 63 401 399 With 86 1.5 417 415 With 87 0.68 363 361 BUT 88 0.71 444 442 BUT 89 0.91 457 455 BUT 90 0.9 411 409 BUT 91 1.05 478 476 BUT 92 1.85 426 424 With 93 1.77 427 425 With 94 1.99 479 477 With 95 1.37 405 403 With 96 1.77 428 426 With 97 0.88 412 470 [M+CH3COO]- BUT 98 1.7 442 440 With 99 1.2 376 374 With 100 1.72 443 441 With

- 107 039788

101 0.99 429 427 A 102 1, 01 420 418 A 103 1, 03 430 428 A 104 1.52 413 411 C 105 1.86 442 440 C 106 1, 67 411 409 C 107 1, 04 445 443 A 108 0.95 437 435 A 109 1.9 398 396 C 110 0.86 417 415 A 111 1, 04 440 438 A 112 1, 67 429 427 C 113 1, 03 443 441 A 114 one 425 423 A 115 1, 02 418 416 A 116 0.76 413 411 A 117 1.79 382 380 C 118 1, 01 442 440 A 119 1.96 442 440 C 120 1.78 431 429 C 121 one 472 470 A 122 2 445 443 C 123 one 400 398 A 124 0.78 415 413 A 125 1.78 453 451 C 126 0.95 392 390 A 127 1, 61 427 425 C 128 eighteen 438 436 C 129 1.95 438 436 C 130 0.94 436 434 A 131 0.88 411 409 A 132 0.93 455 453 A 133 0.93 468 466 A 134 1.78 437 435 C

- 108 039788

135 1, 03 430 428 BUT 136 1.99 418 416 With 137 0.93 401 399 BUT 138 0.88 416 474 [M+CH3COO]~ BUT 139 1.43 376 374 With 140 1.98 438 436 With 141 0.81 402 400 BUT 142 1.32 388 386 With 143 0.77 401 — BUT 144 1.94 472 470 With 145 0.89 430 428 BUT 146 1.32 439 437 With 147 1.88 426 424 With 148 1.89 458 456 With 149 1.72 408 406 With 150 1.38 388 386 With 151 1.41 438 436 With 152 1.56 377 375 With 153 1.70 451 449 With 154 1, 67 407 405 With 155 1.47 391 389 With 156 1.46 377 375 With 157 1, 60 377 375 With 158 1.33 394 392 With 159 1.65 421 419 With 160 1.57 377 375 With 161 1.76 445 443 With 162 1.36 388 386 With 163 1.52 398 396 With 164 1.32 376 374 With 165 2.04 459 456 With 166 1.43 404 402 With 167 1.73 441 439 With

- 109039788

168 0.75 406 404 A 169 1.98 474 472 With 170 1, 83 461 459 With 171 1.96 459 457 With 172 1.86 455 453 With 173 1.35 423 421 With 174 1.58 417 415 With 175 1.77 439 437 With 176 1.52 413 411 With 177 1.36 376 374 With 178 1.45 426 424 With 179 1.46 416 414 With 180 1.88 412 410 With 181 1.32 388 386 With 182 1.73 459 457 With 183 1.54 413 411 With 184 1.50 377 375 With 185 0.83 423 421 BUT 186 0.76 406 404 BUT 187 1.79 413 411 With 188 1, 62 427 425 With 189 100 422 420 BUT 190 0.87 415 413 BUT 191 0.93 440 438 BUT 192 0.96 456 454 BUT 193 0.96 456 454 BUT 194 1.93 458 456 With 195 0.88 420 418 BUT 196 0.81 403 401 BUT 197 0.86 397 395 BUT 198 0.69 390 388 BUT 199 0.66 405 405 AT 200 0.88 457 BUT

- 110 039788

201 0.92 412 470 [M+CH3COO]- A 202 0.76 362 A 203 0.76 455 455 A 204 0.76 455 A 205 0.89 412 A 206 100 400 A 207 0.65 390 448 [M+CH3COO]- A 208 0.93 392 A 209 0.91 412 A 210 0.75 401 A 211 0.75 401 A 212 0.69 412 A 213 0.91 392 390 A 214 0.99 400 398 A 215 0.92 457 A 216 1.20 376 374 C 217 1.79 382 380 C 218 1.90 398 396 C 219 0.94 443 441 A 220 (.HCl) 0.78 401 A 221 0.89 412 410 A 222 0.90 405 403 A 223 0.94 405 403 A 224 0.79 395 393 A 225 1.52 385 383 C 226 1, 11 452 450 A 227 1.60 429 427 C 228 0.92 429 427 A 229 0.87 387 385 A 230 1.37 405 403 C 231 0.64 413 411 A

- 111 039788

232 1.46 388 386 With 233 1.37 388 386 With 234 1.51 398 396 With 235 1.53 407 405 With 236 1.25 390 388 With 237 1.54 408 406 With 238 1, 62 432 430 With 239 1, 63 441 439 With 240 1.38 362 360 With 241 1.93 474 472 With 242 1.85 475 473 With 243 1.49 363 361 With 244 1.36 388 386 With 245 1, 81 426 424 With 246 1.65 431 429 With 247 1.90 470 468 With 248 1.28 374 372 With 249 1.32 374 372 With 250 1.53 391 389 With 251 1.55 391 389 With 252 1.57 391 389 With 253 1.65 441 439 With 254 1.53 391 389 With 255 1.51 406 404 With 256 1.34 374 372 With 257 1.38 374 372 With 258 1.93 419 417 with 259 2.35 484 482 with 260 1.51 377 375 with 261 1.36 389 387 with 262 1.51 391 389 with 263 1.78 455 453 with 264 1.54 407 405 with 265 1.65 427 425 with

- 112 039788

266 2.41 485 483 With 267 0.91 456 454 BUT 268 1, 67 441 439 With 269 0.90 431 429 BUT 270 0.85 439 437 BUT 271 0.78 405 403 BUT 272 0.73 417 415 BUT 273 1.98 438 436 With 274 1.73 439 437 With 275 1.73 417 415 With 276 1, 06 461 459 BUT 277 0.97 449 507 [M+CH3COO]- BUT 278 1.01 445 443 BUT 279 0.97 445 443 BUT 280 1.16 483 481 BUT 281 1.89 453 451 With 282/387 0.93 435 433 BUT 283 1.75 445 443 With 284 5, 27 433 431 BUT 285 1.76 467 465 With 286 1.76 467 465 With 287 1.79 453 451 With 288 0.78 431 429 BUT 289 1, 01 425 423 BUT 290 0.87 429 427 BUT 291 0.92 423 421 BUT 292 442., 83 440 42 C 293 0.95 465 463 BUT 294 0.82 449 447 BUT 295 0.9 437 435 BUT 296 0.94 468 466 BUT 297 0.91 437 435 BUT 298 0.86 463 461 BUT

- 113 039788

299 0.92 437 435 BUT 300 0.98 437 435 BUT 301 0.89 445 443 BUT 302 0.98 412 410 BUT 303 0.79 439 437 BUT 304 0.92 425 423 BUT 305 0.76 439 437 BUT 306 0.79 439 437 BUT 307 0.99 437 435 BUT 308 0.87 435 433 BUT 309 0.81 453 451 BUT 310 0.87 469 467 BUT 311 0.75 435 433 BUT 312 0.82 453 431 BUT 313 0.8 435 433 BUT 314 0.75 435 433 BUT 315 0.76 435 433 BUT 316 1, 08 435 - E 317 0.76 435 433 BUT 318 1.15 435 - E 319 0.97 434 432 BUT 320 0.94 434 432 BUT 321 0.88 437 435 BUT 322 0.75 423 421 BUT 323 0.87 423 421 BUT 324 0.81 423 481 [M+CH3SOSG] BUT 325 0.82 441 439 BUT 326 0.75 423 421 BUT 327 0.9 425 423 BUT 328 0.91 425 483 [M+CH3COO^] BUT 329 0.88 457 515 [M+CH3SOSG] BUT

- 114 039788

331 0.98 422 480 [M+CH3COO^] BUT 332 0.83 441 499 [M+CH3SOSU] BUT 333 1.01 452 450 BUT 334 1, 03 440 438 BUT 335 1.7 458 456 With 336 0.93 448 446 BUT 337 0.9 420 418 BUT 338 100 470 468 BUT 339 0.99 432 430 BUT 340 0.98 452 450 BUT 341 0.9 420 418 BUT 342 1, 02 486 484 BUT 343 1.72 432 430 With 344 1, 87 434 432 With 345 1.8 935 [2M+1] 466 With 346 one 430 428 BUT 347 0.9 404 402 BUT 348 1, 04 430 428 BUT 349 0.91 404 402 BUT 350 0.98 464 462 BUT 351 0.97 424 422 BUT 352 1.02 490 488 BUT 353 1.74 424 422 With 354 0.83 388 386 BUT 355 1.96 465 463 With 356 1, 01 452 450 BUT 357 1.91 470 468 358 1, 83 418 416 With 359 1, 81 450 448 With 360 0.74 430 428 BUT 361 0.68 417 415 BUT

-115 039788

362 1.79 427 425 With 363 1, 84 451 449 With 364 0.8 444 442 BUT 365 1, 64 413 411 With 366 0.75 413 411 BUT 367 0.7 416 414 BUT 368 0.82 455 453 BUT 369 one 432 430 BUT 370 0.97 442 440 BUT 371 0.96 424 422 BUT 372 0.94 440 438 BUT 373 eighteen 451 449 With 374 0.94 455 453 BUT 375 1, 01 432 430 BUT 376 0.92 448 446 BUT 377 0.95 436 434 BUT 378 0.88 455 453 BUT 379 0.7 438 436 BUT 380 0.78 438 436 BUT 381 0.71 436 434 BUT 382 0.74 438 436 BUT 383 0.85 450 448 BUT 384 0.88 457 455 BUT 385 0.9 455 453 BUT 386 1.55 431 429 With 282/387 0.93 435 433 BUT 390 0.99 417 415 E 391 1.25 411 409 E 392 1, 01 461 459 BUT 394 1, 01 473 471 BUT 396 1.88 473 471 With 398 0.99 435 433 BUT 400 0.78 431 429 BUT 402 0.62 417 415 BUT 403 0.9 455 453 BUT 404 0.93 451 449 BUT 405 0.91 451 449 BUT 406 0.75 438 436 BUT

SFC-MS methods.

SFC measurements were performed using a Supercritical Fluid Chromatography (SFC) analytical system equipped with a two-component mixture pump for carbon dioxide (CO ₂ ) and modifier delivery, an automatic dispenser, a column oven, a diode array detector equipped with a flow cell for operation under high pressure, withstand values not exceeding 400 bar. When equipped with a mass spectrometer (MS), the flow from the column was directed to (MS). It is within the purview of one skilled in the art to set tunable parameters (eg, scan range, exposure time, etc.) to obtain ions to determine the nominal monoisotopic molecular weight (MW) of the compound. Data collection and processing was carried out using the appropriate software.

Table 4A. Analytical Methods SFC-MS (flow rate expressed in ml/min;

column temperature (T) in °C; analysis time in minutes, back pressure (BPR) in bar)

the code WAYS Speaker mobile phase Gradient Flow rate T columns Analysis time BPR one Daicel Chiralpak® AD-H column (5.0 µm, 250 χ 4.6 mm) A: CO ₂ B: EtOH+0.2% iPrNH ₂ 40% B, retention 4 min., up to 50% in 1 min. , holding 2 min. 5 7 40 110

- 117 039788

2 Daicel column Chiralpak® AD-3 (3.0 µm, 150 x 4.6 mm) A: CO ₂ B: EtOH+O.2% iPrNH2 from 10% to 50% B for 6 min. , holding 3.5 min. 2.5 9.5 40 110 3 Daicel Chiralpak® AD-H column (5.0 µm, 250 χ 4.6 mm) A: C0 ₂ B: (EtOHiPrOH)+0.2% iPrNH2 35% B, retention 4 min., up to 50% in 1 min. , holding 2 min. 5 7 40 110 4 Daicel column Chiralpak® AD-H (5.0 µm, 250 x 4.6 mm) A: C0 ₂ B: iPrOH+0.2% iPrNH ₂ 25% B, hold 4 min., up to 50% in 1 min. , holding 2 min. 5 7 40 110 5 Daicel Chiralpak® AD-H column (5.0 µm, 250 χ 4.6 mm) A: CO ₂ B: iPrOH+0.2% iPrNH ₂ 30% B, hold 4 min., up to 50% in 1 min. , holding 2 min. 5 7 40 110 6 Daicel Chiralpak® AD-H column (5.0 µm, 250 χ 4.6 mm) A: CO ₂ B: iPrOH+0.2% iPrNH ₂ 40% B, retention 4 min., up to 50% in 1 min. , holding 2 min. 5 7 40 110

- 118 039788

7 Daicel Chiralpak® column AD-3 (3.0 µm, 150 x 4.6 mm) A: C0 ₂ B: iPrOH+0.2% iPrNH ₂ from 10% to 50% B for 6 min. , holding 3.5 min. 2.5 9.5 40 110 eight Daicel Chiralpak® AD-H column (5.0 µm, 250 χ 4.6 mm) A: CO ₂ B: (MeOHiPrOH)+0.2% iPrNH ₂ 25% B, hold 4 min., up to 50% in 1 min. , holding 2 min. 5 7 40 110 nine Daicel Chiralpak® AD-H column (5.0 µm, 250 χ 4.6 mm) A: CO ₂ B: (MeOHiPrOH)+0.2% iPrNH ₂ 30% B, retention 4 min., up to 50% in 1 min. , holding 2 min. 5 7 40 110 ten Daicel column (AD, OD, OJ, AS,)-H (5.0 µm, 250 χ 4.6 mm) A: CO ₂ B: 3 different solvents Λ used for B: (MeOH, EtOH, iPrOH) +0.2% iPrNH ₂ 10% to 40% B in 19.4 minutes, 40 to 50% in 2.00 minutes. , retention 3.6 min. 3 25 thirty 110

- 119 039788

thirteen Speaker Daicel AS-H (5.0 µm, 500 x 4.6 mm) A: CO ₂ B: 3 different solvents L used for B: (MeOH, EtOH, iPrOH) + 0.2% iPrNH ₂ 10% to 40% B in 19.4 minutes, 40 to 50% in 2.00 minutes. , retention 3.6 min. 3 25 fifty 110 12 Speaker Daicel AS-H (5.0 µm, 500 x 4.6 mm) A: CO ₂ B: 3 different solvents L used for B: (MeOH, EtOH, iPrOH) + 0.2% iPrNH ₂ 20% B, 17.5 min retention, 20 to 50% in 3.00 min. , retention 4.1 min. 3 25 fifty 110 thirteen Speaker Daicel OJ-H (5.0um, 250x4.6mm) A: CO ₂ B: 3 different solvents L used for C: MeOH + 0.2% iPrNH ₂ 12% B, hold 10 min. 3 ten thirty 110 fourteen Daicel column Chiralpak® AD-H (5.0 µm, 250 x 4.6 mm) A: CO ₂ B: MeOH + 0.2% iPrNH ₂ 20% B, hold 4 min., up to 50% in 1 min. , holding 2 min. 5 7 40 110

- 120 039788

fifteen Daicel Chiralpak® OD-H column (5.0 µm, 250 χ 4.6 mm) A: CO ₂ B: EtOH+O.2% iPrNH ₂ 30% B, hold 4 min., up to 50% in 1 min. , holding 2 min. 5 7 40 110 sixteen Daicel Chiralpak® OD-3 column (3.0 µm, 150 x 4.6 mm) A: CO ₂ B: EtOH+O.2% iPrNH ₂ from 10% to 50% B for 6 min. , holding 3.5 min. 2.5 9.5 40 110 17 Daicel Chiralpak® AD3 column (3.0 µm, 150x4.6 mm) A: CO ₂ B: EtOH+O.2% iPrNH ₂ from 10% to 50% B for 6 min. , holding 3.5 min. 2.5 9.5 40 130 eighteen Daicel Chiralpak® IC3 column (3.0 µm, 150x4.6 mm) A: CO2 AT: EtOH+O.2% iPrNH2 from 10% to 50% B for 6 min. , holding 3.5 min. 2.5 9.5 40 130

Table 4B. Analytical data SFC-Rt means retention time (in minutes), [M+H]+ means mass of protonated compound, method means the method used to analyze enantiomerically pure compounds using (SFC)MS

CONN. NO. R _T (min.) [M+N] ⁺ Way Elution order 1a 3, 66 365 fourteen Enantiomer 2 1b 2.51 365 fourteen Enantiomer 1 Behind 3 365 eight Enantiomer 1

- 121 039788

sz 2.11 365 eight Enantiomer 2 24a 1.05 445 6 Enantiomer 1 24b 1.52 445 6 Enantiomer 2 25a 4.79 441 sixteen Enantiomer 1 25b 5.48 absence e answer sixteen Enantiomer 1 28 3.95 439 2 Enantiomer 2 29 1.33 413 fifteen Enantiomer 2 thirty 2.54 413 fifteen Enantiomer 1 33 5.45 447 7 Enantiomer 1 34 4.85 447 7 Enantiomer 2 45 1.2 443 4 Enantiomer 2 46 1.56 443 4 Enantiomer 1 62 3.42 417 2 73 2.47 448 5 Enantiomer 1 75 1.42 448 5 Enantiomer 2 81 2.81 419 2 Enantiomer 1 82 3.25 419 2 Enantiomer 2 86 3, 09 417 2 Enantiomer 2 95 6, 8 405 7 Enantiomer 2 97 5, 22 412 thirteen N. d. 99 3.33 376 3 Enantiomer 2 104 1, 67 413 one Enantiomer 2 109 2.54 398 one N. d. 116 2.12 382 nine Enantiomer 2 123 5, 72 400 ten N. d. 140 4, 67 438 2 Enantiomer 2 201 8.79 412 eleven Enantiomer 1 203 9, 22 455 12 Enantiomer 1 204 7.53 455 12 Enantiomer 1 216 2.2 376 3 Enantiomer 1 217 1.29 382 nine Enantiomer 1 218 1.33 398 one Enantiomer 1 230 6, 35 405 7 Enantiomer 2 273 4.21 438 2 Enantiomer 2 274 4.5 439 2 285 6, 81 467 eighteen Diastereomer 4 286 4.95 467 eighteen Diastereomer 3 392 2, 64 461 17 Enantiomer 1

Nuclear magnetic resonance (NMR).

^! H NMR spectra were recorded either on a Bruker DPX-400 spectrometer with standard pulse sequences operating at 400 MHz, or on a Bruker DPX-360 operating at 360 MHz using DMSO-dg (deuterated DMSO, dimethyl-bb-sulfoxide) in as a solvent.

- 122039788

Chemical shift values (δ) are given in parts per million (ppm) relative to tetramethylsilane (TMS), which was used as an internal standard.

Comm. No. 25a: 1H NMR (400 MHz, DMSO-d ₆ at 100 C) δ ppm 1.74-1.93 (m, 4H), 1.97-2.11 (m, 3H), 2.31- 2.45 (m, 1H), 4.06-4.23 (m, 1H), 4.61 (br s, 1H), 4.82 (br s, 1H), 7.02 (t, J= 54.2 Hz, 1H), 7.50 (s, 1H), 7.78 (dd, J=8.4, 1.8 Hz, 1H), 8.17 (d, J=8.4 Hz, 1H), 8.44 (br s, 1H), 8.47 (d, J=1.2 Hz, 1H), 9.45 (s, 1H).

Comm. #93: 1H NMR (400 MHz, DMSO-d6 at 100 C) δ ppm 0.84 (d, J=6.6 Hz, 3H), 0.88-0.98 (m, 4H), 1, 45 (qd, J=12.4, 4.4 Hz, 1H), 1.91 (br dd, J=13.4, 2.6 Hz, 1H), 2.06-2.14 (m, 1H ), 2.42 (s, 3H), 2.45-2.49 (m, 1H), 3.13 (br t, J=11.6 Hz, 1H), 3.37 (dd, J=12 .9, 11.1 Hz, 1H), 3.60 (td, J=10.9, 4.0 Hz, 1H), 4.17 (br s, 2H), 6.79-7.23 (m , 3H), 7.57 (s, 1H), 8.72 (s, 1H);

Comm. #108: 1H NMR (400 MHz, DMSO-d6) δ ppm 0.77 (d, J=6.4 Hz, 3H), 1.34-1.56 (m, 1H), 1.68-2, 00 (m, 1H), 2.43 (br s, 1H), 2.76-3.28 (m, 2H), 3.49-3.74 (m, 2H), 3.87 (s, 3H ), 4.46-4.76 (m, 1H), 6.81-7.33 (m, 3H), 7.65 (s, 1H), 8.80 (s, 1H);

Comm. #110: 1H NMR (400 MHz, DMSO-d6 at 100 C) δ ppm 0.83 (d, J=6.6 Hz, 3H), 1.39-1.50 (m, 1H), 1, 85-1.95 (m, 1H), 2.43 (s, 3H), 2.44-2.48 (m, 1H), 3.13 (br t, J=12.3 Hz, 1H), 3.37 (dd, J=13.0, 11.2 Hz, 1H), 3.58 (td,

J=10.9, 4.0 Hz, 1H), 3.88 (s, 3H), 4.13 (br s, 2H), 6.58 (s, 1H), 6.82 (s, 1H) , 7.01 (t, J=54.2 Hz, 1H), 7.56 (s, 1H), 8.71 (s, 1H);

Comm. No. 132: 1H NMR (360 MHz, DMSO-d ₆ ) δ ppm 0.78 (d, J=6.6 Hz, 3H), 1.38-1.61 (m, 1H), 1.67-2 .01 (m, 1H), 2.60 (s, 3H), 2.86-3.30 (m, 2H), 3.49 (br d, J=14.6 Hz, 1H), 3.59 -3.72(m, 2H), 4.48-4.79(m, 1H), 6.877.35(m, 1H), 7.54-7.89(m, 3H), 8.79(s , 1H);

Comm. #228: 1H NMR (400 MHz, DMSO-d ₆ at 100 C) δ ppm 0.85 (d, J=6.6 Hz, 4H), 1.43-1.56 (m, 1H), 1 .86-1.99 (m, 1H), 3.19 (td, J=12.9, 2.8 Hz, 1H), 3.42 (dd, J=13.0, 11.2 Hz, 1H ), 3.64 (td, J=10.9, 4.0 Hz, 1H), 4.15-4.26 (m, 1H), 4.28-4.38 (m, 1H), 7, 01 (t, J=54.2 Hz, 1H), 7.58 (br s, 1H), 7.61 (dd, J=8.4, 1.5 Hz, 1H), 8.11 (d, J=7.9Hz, 1H), 8.27(d, J=1.3Hz, 1H), 8.70(s, 1H), 9.40(s, 1H);

Comm. #281: 1H NMR (400 MHz, DMSO-d6 at 120 C) δ ppm 0.82 (d, J=6.40 Hz, 3H), 1.46 (qd, J=12.47, 4.40 Hz, 1H), 1.84-1.93 (m, 1H), 2.41-2.53 (m, 1H), 2.84 (s, 3H), 3.07-3.18 (m, 1H), 3.35 (dd, J=12.98, 11.22Hz, 1H), 3.58 (td, J=10.95, 3.85Hz, 1H), 3.88-4.33 (m, 2H), 6.85 (s, 1H), 6.98 (t, J=54.36 Hz, 1H), 7.12 (s, 1H), 7.53 (s, 1H), 7 .55 (t, J=73.07 Hz, 1H), 7.74 (s, 1H), 8.67 (s, 1H);

Comm. #295: 1H NMR (400 MHz, DMSO-d6 at 100 C) δ ppm 1.67-1.87 (m, 4H), 1.89-2.10 (m, 3H), 2.26-2 .38 (m, 1H), 3.82 (s, 3H), 4.13 (br d, J=12.10 Hz, 1H), 4.56 (br s, 1H), 4.86 (br s , 1H), 6.55 (d, J=2.64 Hz, 1H), 7.02 (t, J=55.00 Hz, 1H), 7.35 (d, J=3.08 Hz, 1H ), 7.41-7.49 (m, 3H), 7.90 (s, 1H), 8.59 (s, 1H);

Comm. #321: 1H NMR (400 MHz, DMSO-d ₆ at 100 CELSIUS) δ ppm 0.81 (d, J=6.60 Hz, 3H), 1.47 (qd, J=12.43, 4, 29 Hz, 1H), 1.85-1.93 (m, 1H), 2.39-2.46 (m, 1H), 2.94 (s, 3H), 3.09-3.18 (m , 1H), 3.33-3.40 (m, 1H), 3.57-3.64 (m, 1H), 3.83-4.52 (m, 2H), 6.82 (m, 1H ), 7.00 (m, 1H), 7.43 (s, 1H), 7.46 (s, 1H), 7.55 (s, 1H), 8.69 (s, 1H);

Comm. #335: 1H NMR (400 MHz, DMSO-d ₆ at 100 C) δ ppm 1.74-2.00 (m, 6H), 2.02-2.08 (m, 1H), 2.36- 2.41 (m, 1H), 2.42 (s, 3H), 3.84 (s, 3H), 4.08-4.14 (m, 1H), 4.81-4.85 (m, 1H), 5.135.17 (m, 1H), 7.05 (t, J=54.40 Hz, 1H), 7.56 (s, 1H), 7.89 (s, 1H), 8.69 ( s, 1H);

Comm. #356: 1H NMR (400 MHz, DMSO-d ₆ at 100 C) δ ppm 1.68-1.89 (m, 4H), 1.92-2.04 (m, 3H), 2.30- 2.45 (m, 1H), 4.01-4.07 (m, 1H), 4.39-4.84 (m, 2H), 7.01 (t, J=52.80 Hz, 1H) , 7.49 (s, 1H), 7.58 (dd, J=8.25, 1.87 Hz, 1H), 7.71 (d, J=8.14 Hz, 1H), 7.83 ( d, J=1.54 Hz, 1H), 8.58 (brs, 1H).

Pharmacological examples

The compounds provided in the present invention are inhibitors of PDE2, in particular PDE2A. The results of testing the compounds in several pharmacological assays are shown below.

PDE2A in vitro analysis

Recombinant human PDE2A (hPDE2A) was expressed in Sf9 cells using a recombinant baculovirus-based rPDElOA construct. Cells were harvested 48 hours post-infection and hPDE2A protein was purified by metal chelate chromatography on Ni-sepharose 6FF. Test compounds were dissolved and diluted in 100% DMSO to 100 times the final concentration in the assay. Compound dilutions (0.4 μl) were added to 384-well plates to 20 μl of incubation buffer (50 mM Tris, pH 7.8, 8.3 mM MgCl ₂ , 1.7 mM EGTA). 10 μl of hPDE2A enzyme was added to the incubation buffer and the reaction was started by adding 10 μl of substrate to a final concentration of 10 μM cGMP and 0.01 μCi ³ H-cGMP. The reaction mixture was incubated for 45 min at room temperature. After incubation, the reaction was stopped with 20 μl of a stop reagent consisting of PDE beads for SPA (scintillation proximity assay) at a concentration of 17.8 mg/ml, supplemented with 200 mm ZnCl ₂ . After pellets had settled for 30 minutes, radioactivity was measured using a Perkin Elmer Topcount scintillation counter and the results were expressed in cpm. To obtain blank values, the enzyme was omitted from the reaction mixture and replaced with incubation buffer. Control values were obtained by adding

- 123 039788

DMSO at a final concentration of 1% instead of compound. A best fit curve was fitted point by point using the least sum of squares method on a plot of % control minus blank versus compound concentration, and from this curve a half-maximal inhibition concentration (IC50) was obtained.

PDE3A in vitro analysis

Recombinant human PDE3A (hPDE3A) was purchased as partially purified insect cell lysate from Scottish Biomedical, cloned from human brain and expressed in Sf9 cells. Test compounds were dissolved and diluted in 100% DMSO to 100 times the final concentration in the assay. Compound dilutions (0.4 μl) were added to 384-well plates to 20 μl of incubation buffer (50 mM Tris, pH 7.8, 8.3 mM MgCl ₂ , 1.7 mM EGTA). 10 μl hPDE3A enzyme was added to the incubation buffer and the reaction started by adding 10 μl substrate to a final concentration of 0.4 μM cAMP and 2.4 μCi/ml [ ³ H]-cAMP. The reaction mixture was incubated for 60 min at room temperature. After incubation, the reaction was stopped with 20 μl of a stop reagent consisting of PDE beads for SPA (scintillation proximity assay) at a concentration of 17.8 mg/ml, supplemented with 200 mm ZnCl ₂ . After sedimentation of the pellets for 30 minutes, the radioactivity was measured with a Perkin Elmer Topcount scintillation counter and the results were expressed in cpm. To obtain blank values, the enzyme was omitted from the reaction mixture and replaced with incubation buffer. Control values were obtained by adding DMSO at a final concentration of 1% in place of the compound. A best fit curve was fitted point by point using the least sum of squares method on a plot of % control minus blank versus compound concentration, and from this curve a half-maximal inhibition concentration (IC50) was obtained.

PDE10A in vitro assay

Recombinant rat PDE10A (rPDE10A2) was expressed in Sf9 cells using a recombinant baculovirus-based rPDElOA construct. Cells were harvested 48 hours post-infection and rPDElOA protein was purified by metal chelate chromatography on Ni-Sepharose 6FF. Test compounds were dissolved and diluted in 100% DMSO to 100 times the final concentration in the assay. Recombinant human PDE10A (hPDE2A) was expressed in Sf9 cells using a recombinant baculovirus hPDElOA construct that was generated and amplified in the laboratory. Cells were harvested 72 hours post-infection and the hPDElOA protein was purified by metal chelate chromatography on Ni-Sepharose. Compound dilutions (0.4 μl) were added to 384-well plates to 20 μl of incubation buffer (50 mM Tris, pH 7.8, 8.3 mM MgCl2, 1.7 mM EGTA). 10 μl of rPDElOA or hPDElOA enzyme was added to the incubation buffer and the reaction was started by adding 10 μl of substrate to a final concentration of 60 nM cAMP and 0.008 μCi ³ H-cAMP. The reaction mixture was incubated for 60 min at room temperature. After the incubation, the reaction was stopped with 20 µl stop reagent consisting of PDE pellets for SPA (scintillation proximity assay) at a concentration of 17.8 mg/ml. After sedimentation of the pellets for 30 minutes, the radioactivity was measured with a Perkin Elmer Topcount scintillation counter and the results were expressed in cpm. To obtain blank values, the enzyme was omitted from the reaction mixture and replaced with incubation buffer. Control values were obtained by adding DMSO at a final concentration of 1% in place of the compound. A curve of best fit was fitted point by point using the least sum of squares method on a plot of % control minus blank versus compound concentration, and from this curve a half-maximal inhibition concentration (IC ₅₀ ) was obtained.

Tab. 5: in vitro data for the compounds of the present invention. By default, PDE10A inhibition data refer to the human clone (also denoted by (h)), unless indicated by (r), referring to the rat clone.

- 124 039788

Table 5

No. conn. pIC ₅₀ hPDE2A p ^b hPDE2A pIC ₅₀ hPDE3B π ^{1 b} hPDE3 B pIC ₅₀ hPDE10A2 or rPDE10A2 π ¹ max hPDE10A2 or rPDE10A2 1b 5.96 88 <5 thirteen 5, 01 fifty la 9, 1 100 5, 68 65 7, 89 98 2b 7.72 100 <5 31 6.57 95 2a 9, 23 101 6, 11 98 8.14 101 3b 5, 25 64 <5 fourteen <5 nineteen 3a 8.24 97 5.46 77 6.57 93 4 8, 84 100 5, 24 70 7, 62 99 5 9, 02 100 6, 23 97 8, 05 100 6 8.79 99 6, 17 97 7.75 100 7 9, 1 100 7, 12 100 7.77 101 eight 8.96 100 6.56 97 7.95 100 nine 7.86 101 5, 37 74 7, 13 100 ten 8.96 100 7, 14 101 8.34 100 eleven 7, 64 100 5, 32 69 6, 43 95 12 7, 67 101 5.96 94 6, 44 97 thirteen 7.98 100 <5 38 6.46 95 fourteen 8.22 99 5, 33 70 7, 05 99 fifteen 8.49 100 5, 09 54 7, 14 97 sixteen 8.21 100 5.78 88 7.2 98 17 8.31 99 5, 37 74 7, 09 97 eighteen 8.33 100 5, 19 65 6.71 97 nineteen 7.98 101 5.51 79 6, 89 96 20 7.92 101 5, 37 74 6, 88 98 21 8.34 99 5, 82 89 6, 88 97 22 8, 63 100 5, 82 87 7, 8 99 23 9, 33 101 5.98 90 8.15 100 24a 5.59 80 <5 31 <5 23 24b 8.19 98 <5 28 5, 13 60 25a 9, 15 100 5, 04 57 6.41 96 25b 5.96 92 <5 37 <5 28

- 125 039788

26a <5 21 <5 0 <5 7 26b 8, 17 98 <5 41 6, 63 95 27 9, 04 100 <5 37 5.96 88 28 9, 02 101 <5 17 6, 35 95 29 8, 82 101 <5 eighteen 6.16 90 thirty 5.45 77 <5 eight <5 32 31 8, 6 100 <5 eight 5.95 84 32 8.46 100 <5 31 5, 84 81 33 7.83 99 <5 25 5.21 67 34 5.71 88 <5 31 <5 28 35 8, 17 100 <5 one 5.59 76 38 8.21 100 <5 43 5, 64 80 39 8, 08 98 <5 sixteen 5.51 75 40 9.45 101 5, 15 58 6.91(h) 7.1(r) 97(h) 96(r) 41 7.86 99 <5 17 5, 38 72 42 8.94 100 <5 48 6, 5 95 43 8.3 99 <5 29 5, 88 88 44 8, 02 100 <5 39 5.59 79 45 <5 37 <5 one <5 24 46 7.82 100 <5 5 5.65 86 47 8, 6 99 5, 47 80 6, 19 92 48 9.76 100 7, 06 99 7.36 100 49 8, 68 99 5, 24 42 6.29 95 fifty 8.51 100 <5 61 6, 14 93 51 8.26 101 <5 44 5, 9 86 52 7.86 101 <5 22 5.52 76 53 8.25 100 5, 17 62 6, 11 89 54 8.38 100 5, 02 51 6, 18 89 55 7.9 101 <5 41 5.75 83 56 7.3 101 <5 38 5, 25 63 57 7.56 100 <5 21 5, 5 78 58 7, 61 99 <5 eighteen 5, 35 69 59 8.43 98 5, 07 56 6, 23 95

- 126 039788

60 9, 07 100 5.96 92 6, 82 97 61 8.36 101 <5 45 6, 34 92 63 8, 14 100 5.16 60 6, 06 89 64 8, 8 99 5, 17 62 6.45 96 65 8.9 100 5, 17 70 6, 6 95 66 7.76 99 <5 48 5, 64 79 67 8.59 99 <5 49 6.54 94 68 8.2 99 5 fifty 5.86 85 69 9, 39 102 6, 42 95 7.34 100 70 8.76 102 5.21 63 6, 42 95 71 8.73 98 <6 32 6, 47 94 72 8.99 100 <6 46 6.98 92 73 8.71 101 5.58 83 6, 66 98 74 7.52 101 <5 thirty <5 36 75 5, 53 81 <5 22 <5 27 76 9, 64 99 5, 91 91 7.5 101 77 9, 34 101 6, 17 96 7.08 98 78 nine 102 5, 8 87 6.98 98 79 8.76 99 6, 04 93 6, 72 97 80 9, 1 100 5, 38 71 7.09 98 81 5, 15 59 <5 17 <5 one 82 7.47 101 <5 nine <5 eighteen 83 9, 03 100 5, 1 58 7.01 98 84 8.5 101 5, 13 thirty 6, 15 89 85 8, 19 100 5, 11 37 5, 92 88 87 6, 67 98 <5 eleven 5, 34 69 88 9, 68 101 6.41 97 8.4 99 89 9, 64 104 6, 84 96 7.68(h) 7.92 (g) 101(h) 98 (g) 90 9.45 101 6, 67 98 7.71 102 91 9, 43 100 6, 15 61 7, 65 94 92 9, 4 100 5, 88 88 7, 61 98 93 9, 4 101 5, 61 84 7.44 99 94 9, 3 101 6.98 99 7.93 97

- 127 039788

95 9.29 100 5 55 7.32 98 96 9.26 100 6, 4 73 7.48 95 97 9, 24 101 5, 89 93 7.27(h) 7.46(r) 101(h) 99(r) 98 9, 22 100 6, 22 94 7.4 98 99 9, 22 100 5, 68 87 8, 02 100 100 9, 18 101 6.57 99 7.79 100 101 9, 18 101 5, 32 70 7.24 98 102 9, 17 100 <6 44 7.58 95 103 9, 15 99 7, 06 100 7.74 100 104 9, 09 100 6.29 92 7, 13 98 105 9, 06 101 5, 87 90 7.53 101 106 9, 05 100 6.41 98 7, 67 99 107 9, 04 100 5, 38 61 7, 09 99 108 9, 04 100 5.57 80 7.28 99 109 9, 03 101 6, 34 98 8, 18 99 110 9, 02 101 5.58 78 7, 14 98 111 9, 01 100 5.98 92 7.42 99 112 nine 101 5.75 84 7.32 99 113 nine 99 5.95 88 7.42 99 114 8.98 100 7, 05 98 7.34 101 115 8.98 100 5.71 87 7, 11 99 116 8.94 100 6, 07 97 8, 01 99 116 8.95 101 5, 66 82 7, 1 98 118 8.78 101 6, 02 92 7, 13 99 119 8.76 100 7.21 101 7, 11 99 120 8.76 100 6, 15 95 6, 7 96 121 8.75 99 6.29 95 7.23 96 122 8.73 100 5, 74 86 7, 13 100 123 8.71 99 5.79 87 7, 04 99 124 8, 69 99 6, 38 98 7.44 98 125 8, 68 100 6, 23 96 6, 23 96 126 8, 68 101 5, 22 65 6.95 97 127 8, 65 99 6.36 94 7.59 99

- 128 039788

128 8, 64 100 6, 03 90 6, 81 98 129 8, 64 101 5.71 85 7, 04 99 130 8, 61 100 5.76 87 6, 87 96 131 8, 61 98 5, 11 61 7.39 99 132 8, 6 101 <5 46 6, 7 98 133 8.53 100 5.79 85 6, 87 98 134 8.5 101 5.65 84 6, 8 100 135 8.48 100 5.58 81 6, 72 97 136 8.47 101 <5 51 6.54 97 137 7.55 100 <5 35 5, 87 84 139 7.54 99 <5 20 5, 62 78 140 7.53 100 5, 05 57 5.51 83 141 7.53 100 <5 12 5, 69 81 142 7.52 100 <5 35 5.59 79 143 7.48 97 5, 1 58 5, 62 77 144 7.46 100 5, 62 86 6, 33 96 145 7.44 99 5, 69 86 6, 19 93 147 7.42 100 5.65 81 6, 06 90 148 7.41 101 5, 25 67 6, 04 89 149 7.41 99 5.29 66 5, 74 83 150 7.4 100 <5 22 5.45 72 152 7.38 99 <5 thirty 5, 69 80 153 7.36 100 5.51 76 5.97 89 154 7.29 100 <5 fifty 5.56 79 155 7.27 98 <5 37 5, 53 72 156 7.23 100 <5 nineteen 5, 7 81 157 7.22 99 <5 33 5.59 75 158 7.19 100 <5 nineteen 5, 67 78 159 7, 14 100 <5 42 5.49 75 160 7, 14 99 <5 34 5.99 88 161 7, 13 98 <5 eleven 5.51 74 162 7.13 97 <5 26 5, 37 72 163 7, 05 98 <5 12 5.49 74 164 7, 05 100 <5 22 5.71 82

- 129 039788

165 7, 03 100 <5 36 5.46 78 166 7.03 98 <5 32 5.65 81 167 7, 01 97 5, 27 64 5, 7 81 168 8.4 98 5.29 66 7.27 99 169 8.43 98 5.75 82 6.75 98 170 8.34 100 5, 67 84 5, 89 84 171 7.93 99 5, 94 93 6.45 97 172 8.34 101 6.57 98 7.49 100 173 7.86 101 <5 31 6.52 96 174 8.28 99 5, 88 85 6, 24 88 175 8.31 100 5, 22 71 6.85 98 176 8, 04 100 5, 06 56 6.05 88 177 8.02 99 <5 21 6, 34 93 178 7, 87 101 5, 13 62 6, 1 92 179 7, 67 100 <5 32 6, 27 90 180 8.3 100 5.29 66 6.76 97 181 8.34 98 <5 52 6.51 94 182 7, 6 100 <5 44 6, 38 95 183 7.58 99 <5 36 5, 64 79 184 7.55 100 <5 17 5.56 75 186 7.97 100 5, 22 69 6, 47 95 187 7.75 99 5.46 74 6, 37 95 188 8, 07 99 5, 81 87 6, 68 98 189 8.29 100 5.56 80 6, 73 96 190 7.99 100 <5 39 6, 8 96 191 7, 89 100 5.41 74 6.65 97 192 8, 03 99 6, 14 95 7, 14 99 193 8.23 100 5.46 76 6.86 98 194 7.73 99 5, 34 57 6.58 97 195 8.35 98 5, 25 70 6, 43 93 196 7, 63 99 5, 31 68 6, 25 92 197 7.99 98 <5 42 6, 68 96 198 8.25 98 <5 35 6, 38 91 199 7.27 92 5, 08 62 5.23(h) 62(h)

- 130 039788

5.32 (g) 21(d) 200 7, 01 91 <5 35 <5 thirteen 201 6, 19 61 <5 24 5.51 (g) -1 (d) 202 5.57 thirty <5 eighteen <5(h) <5 (d) 38(h) 4 (g) 203 6, 02 48 <5 41 <5 (d) 14 (g) 204 6.79 88 5, 13 48 <5 (d) 13 (g) 205 8.94 99 5.65 83 7, 01 98 206 5.78 39 <5 -one <5 sixteen 207 5, 1 21 <5 2 <5 -5 208 6, 23 62 <5 thirty <5 4 209 6, 14 57 <5 25 <5 7 210 6.21 56 <5 5 <5 sixteen 211 <5 3 <5 thirteen <5 ten 212 6, 24 61 <5 7 <5 eight 213 5, 63 28 <5 eleven <5 23 214 5, 2 nine <5 nine <5 thirty 215 6.58 82 5 45 5, 13 63 216 6.36 98 <5 26 5, 35 67 217 6, 25 99 <5 37 5, 32 72 218 6.69 100 <5 51 5.65 85 219 6.86 100 <5 eighteen 5, 04 45 220 (.HCl) 6, 01 54 <5 7 <5 27 221 5, 92 45 <5 4 <5 26 222 6.55 98 <5 thirteen 5, 17 59 223 6.71 98 <5 nineteen 5.46 71 226 9, 06 100 6, 89 100 7, 09 100 227 8, 66 101 6, 42 95 7.34 98 228 9, 65 100 5, 92 91 7.77 100 229 6, 25 95 <5 eighteen <5 33 230 5.99 93 <5 40 5, 27 65 231 8, 87 97 5, 04 57 7.71 100 232 6, 67 99 <5 31 <5 48

- 131 039788

233 6, 24 94 <5 ten <5 26 234 6.29 96 <5 22 5, 06 59 235 6, 91 98 <5 32 5.26 67 236 6, 68 100 <5 eighteen 5, 11 58 237 6, 73 97 <5 20 5, 09 51 238 6, 42 94 <5 35 5 48 239 6, 72 96 <5 sixteen <5 43 240 5, 81 90 <5 nine 5, 04 51 241 7, 68 97 5.76 88 6.57 94 242 6, 87 97 5.21 67 5, 09 56 243 6, 88 99 <5 thirteen 5, 44 70 244 6, 17 92 <5 12 5, 03 54 245 6, 91 99 5, 23 62 5, 66 79 246 6, 9 98 <5 25 <5 44 247 6, 81 98 5, 69 85 5, 54 77 248 6.46 95 <5 2 4.98 49 249 6, 43 95 <5 ten <5 45 250 6, 88 99 <5 28 5, 31 64 251 6.55 98 <5 17 5, 06 53 252 6, 15 96 <5 17 5, 17 66 253 6, 34 95 <5 17 <5 39 254 6.97 99 <5 25 5, 42 70 255 6.57 95 <5 28 5, 11 58 256 6.49 97 <5 12 5 48 257 6.58 95 <5 fifteen <5 45 258 6, 82 99 5, 81 89 5, 9 90 259 6, 37 99 5.45 82 5, 77 92 260 6.86 97 <5 23 5, 53 74 261 6, 44 95 <5 nineteen <5 45 262 6, 81 101 <5 37 5.49 74 263 6, 89 99 5, 31 65 5.29 64 264 6.98 100 4.98 47 5.48 74 265 8, 62 102 5, 2 55 7.43 98 266 6, 38 100 5, 27 74 5.58 89

- 132 039788

267 9, 4 102 7.21 100 7.95 99 268 5, 72 82 <5 thirteen <5 39 269 9, 15 101 5, 43 78 6.16 93 270 8, 63 100 <5 29 6.57 96 271 8.53 99 5, 13 61 6.79 95 272 8, 66 102 5, 05 54 6, 1 93 273 8, 63 100 6, 09 96 6, 01 96 274 6.54 97 <5 7 5, 22 63 la 9, 1 100 5, 68 65 7, 89 98 lb 5.96 88 <5 thirteen 5, 01 fifty 2a 9, 23 101 6, 11 98 8, 14 101 2b 7.72 100 <5 31 6.57 95 3a 8.24 97 5.46 77 6.57 93 3b 5, 25 64 <5 fourteen <5 nineteen 4 8.84 100 5, 24 70 7, 62 99 5 9, 02 100 6, 23 97 8, 05 100 6 8.79 99 6, 17 97 7.75 100 7 9, 1 100 7, 12 100 7.77 101 eight 8.96 100 6.56 97 7.95 100 nine 7.86 101 5, 37 74 7, 13 100 ten 8.96 100 7, 14 101 8.34 100 eleven 7, 64 100 5, 32 69 6, 43 95 12 7, 67 101 5.96 94 6, 44 97 thirteen 7.98 100 <5 38 6.46 95 fourteen 8.22 99 5, 33 70 7, 05 99 fifteen 8.49 100 5, 09 54 7, 14 97 sixteen 8.21 100 5.78 88 7.2 98 17 8.31 99 5, 37 74 7, 09 97 eighteen 8.33 100 5, 19 65 6.71 97 nineteen 7.98 101 5.51 79 6, 89 96 20 7.92 101 5, 37 74 6, 88 98 21 8.34 99 5, 82 89 6, 88 97 22 8, 63 100 5, 82 87 7.8 99 23 9, 33 101 5.98 90 8.15 100

- 133 039788

24a 5.59 80 <5 31 <5 23 24b 8, 19 98 <5 28 5, 13 60 25a 8.97 100 5, 06 54 6, 23 94 25b 5.96 92 <5 37 <5 28 26a <5 21 <5 0 <5 7 26b 8, 17 98 <5 41 6, 63 95 27 9, 04 100 <5 37 5.96 88 28 9, 02 101 <5 17 6, 35 95 29 8, 82 101 <5 eighteen 6.16 90 thirty 5.45 77 <5 eight <5 32 31 8, 6 100 <5 eight 5.95 84 32 8.46 100 <5 31 5, 84 81 33 7, 83 99 <5 25 5.21 67 34 5.71 88 <5 31 <5 28 35 8.17 100 <5 one 5.59 76 36 8.56 100 5, 4 64 6, 33 93 37 7.76 100 5, 05 52 6, 37 93 38 8.21 100 <5 43 5, 64 80 39 8, 08 98 <5 sixteen 5.51 75 40 9.45 101 5, 15 58 6.91(h) 7.1(r) 97(h) 96(r) 41 7.86 99 <5 17 5, 38 72 42 8.94 100 <5 48 6, 5 95 43 8.3 99 <5 29 5, 88 88 44 8, 02 100 <5 39 5.59 79 45 <5 37 <5 one <5 24 46 7, 82 100 <5 5 5.65 86 47 8, 6 99 5, 47 80 6, 19 92 48 9.76 100 7, 06 99 7.36 100 49 8, 68 99 5, 24 42 6.29 95 fifty 8.51 100 <5 61 6, 14 93 51 8.26 101 <5 44 5, 9 86 52 7.86 101 <5 22 5.52 76 53 8.25 100 5, 17 62 6, 11 89

- 134 039788

54 8.38 100 5, 02 51 6, 18 89 55 7.9 101 <5 41 5.75 83 56 7.3 101 <5 38 5, 25 63 57 7.56 100 <5 21 5, 5 78 58 7, 61 99 <5 eighteen 5, 35 69 59 8.43 98 5, 07 56 6, 23 95 60 9, 07 100 5.96 92 6, 82 97 61 8.36 101 <5 45 6, 34 92 62 5.52 77 <5 eight <5 36 63 8, 14 100 5.16 60 6, 06 89 64 8, 8 99 5, 17 62 6.45 96 65 8.9 100 5, 17 70 6, 6 95 66 7.76 99 <5 48 5, 64 79 67 8.59 99 <5 49 6.54 94 68 8.2 99 5 fifty 5.86 85 69 9, 39 102 6, 42 95 7.34 100 70 8.71 102 5.29 66 6.48 93 71 8.73 98 <6 32 6, 47 94 72 8.99 100 <6 46 6.98 92 73 8.71 101 5.58 83 6.66 98 74 7.52 101 <5 thirty <5 36 75 5, 53 81 <5 22 <5 27 76 9, 64 99 5, 91 91 7.5 101 77 9, 34 101 6, 17 96 7, 08 98 78 nine 102 5, 8 87 6.98 98 79 8.76 99 6, 04 93 6, 72 97 80 9, 1 100 5, 38 71 7, 09 98 81 5, 15 59 <5 17 <5 one 82 7, 47 101 <5 nine <5 eighteen 83 9, 03 100 5, 1 58 7, 01 98 84 8.5 101 5, 13 thirty 6, 15 89 85 8, 19 100 5, 11 37 5, 92 88 86 7.3 100 <5 34 <5 40 87 6, 67 98 <5 eleven 5, 34 69

- 135 039788

88 9, 68 101 6.41 97 8.4 99 89 9, 64 104 6, 84 96 7.68(h) 7.92(r) 101(h) 98(r) 90 9.45 101 6, 67 98 7.71 102 91 9, 43 100 6, 15 61 7, 65 94 92 9, 4 100 5, 88 88 7, 61 98 93 9, 34 101 5.59 84 7.4 99 94 9, 3 101 6.98 99 7.93 97 95 9.29 100 5 55 7.32 98 96 9.26 100 6, 4 73 7.48 95 97 9, 24 101 5, 89 93 7.27(h) 7.46(r) 101(h) 99(r) 98 9, 22 100 6, 22 94 7.4 98 99 9, 22 100 5, 68 87 8, 02 100 100 9, 18 101 6.57 99 7.79 100 101 9, 18 101 5, 32 70 7.24 98 102 9, 17 100 <6 44 7.58 95 103 9, 15 99 7, 06 100 7.74 100 104 9, 09 100 6.29 92 7, 13 98 105 9, 06 101 5, 87 90 7.53 101 106 9, 05 100 6.41 98 7, 67 99 107 9, 04 100 5, 38 61 7, 09 99 108 9, 04 100 5.57 80 7.28 99 109 9, 03 101 6, 34 98 8, 18 99 110 8, 88 100 5, 47 76 6.95 98 111 9, 01 100 5.98 92 7.42 99 112 nine 101 5.75 84 7.32 99 113 nine 99 5.95 88 7.42 99 114 8.98 100 7, 05 98 7.34 101 115 8.98 100 5.71 87 7.11 99 117 8.94 100 6, 07 97 8, 01 99 116 8.95 101 5, 66 82 7.1 98 118 8.78 101 6, 02 92 7, 13 99 119 8.76 100 7.21 101 7, 11 99

- 136 039788

120 8.76 100 6, 15 95 6, 7 96 121 8.75 99 6.29 95 7.23 96 122 8.73 100 5, 74 86 7, 13 100 123 8.71 99 5.79 87 7, 04 99 124 8, 69 99 6, 38 98 7.44 98 125 8, 68 100 6, 23 96 6, 23 96 126 8, 68 101 5, 22 65 6.95 97 127 8, 65 99 6.36 94 7.59 99 128 8, 64 100 6, 03 90 6, 81 98 129 8, 64 101 5.71 85 7, 04 99 130 8, 61 100 5.76 87 6, 87 96 131 8, 61 98 5, 11 61 7.39 99 132 8, 6 101 <5 46 6, 7 98 133 8.53 100 5.79 85 6, 87 98 134 8.5 101 5.65 84 6, 8 100 135 8.48 100 5.58 81 6, 72 97 136 8.47 101 <5 51 6.54 97 137 7.55 100 <5 35 5, 87 84 138 7.55 93 5, 31 64 <5 24 139 7.54 99 <5 20 5, 62 78 140 7.53 100 5, 05 57 5.51 83 141 7.53 100 <5 12 5, 69 81 142 7.52 100 <5 35 5.59 79 143 7.48 97 5, 1 58 5, 62 77 144 7.46 100 5, 62 86 6, 33 96 145 7.44 99 5, 69 86 6, 19 93 146 7.43 100 5.78 85 5.48 76 147 7.42 100 5.65 81 6, 06 90 148 7.41 101 5, 25 67 6, 04 89 149 7.41 99 5.29 66 5, 74 83 150 7.4 100 <5 22 5.45 72 151 7.39 100 <5 eleven 5.86 85 152 7.38 99 <5 thirty 5, 69 80 153 7.36 100 5.51 76 5.97 89

- 137 039788

154 7.29 100 <5 fifty 5.56 79 155 7.27 98 <5 37 5, 53 72 156 7.23 100 <5 nineteen 5, 7 81 157 7.22 99 <5 33 5.59 75 158 7, 19 100 <5 nineteen 5, 67 78 159 7, 14 100 <5 42 5.49 75 160 7.14 99 <5 34 5.99 88 161 7, 13 98 <5 eleven 5.51 74 162 7, 13 97 <5 26 5, 37 72 163 7.05 98 <5 12 5.49 74 164 7, 05 100 <5 22 5.71 82 165 7, 03 100 <5 36 5.46 78 166 7, 03 98 <5 32 5.65 81 167 7.01 97 5, 27 64 5, 7 81 168 8.4 98 5.29 66 7.27 99 169 8.43 98 5.75 82 6.75 98 170 8.34 100 5, 67 84 5, 89 84 171 7, 93 99 5, 94 93 6.45 97 172 8.34 101 6.57 98 7.49 100 173 7.86 101 <5 31 6.52 96 174 8.28 99 5, 88 85 6, 24 88 175 8.31 100 5, 22 71 6.85 98 176 8, 04 100 5, 06 56 6.05 88 177 8, 02 99 <5 21 6, 34 93 178 7, 87 101 5, 13 62 6, 1 92 179 7, 67 100 <5 32 6, 27 90 180 8.3 100 5.29 66 6.76 97 181 8.34 98 <5 52 6.51 94 182 7, 6 100 <5 44 6, 38 95 183 7.58 99 <5 36 5, 64 79 184 7.55 100 <5 17 5.56 75 185 7, 66 98 5.48 79 5, 92 87 186 7.97 100 5, 22 69 6, 47 95 187 7.75 99 5.46 74 6, 37 95

- 138 039788

188 8, 07 99 5, 81 87 6, 68 98 189 8.29 100 5.56 80 6, 73 96 190 7.99 100 <5 39 6, 8 96 191 7, 89 100 5.41 74 6.65 97 192 8, 03 99 6, 14 95 7, 14 99 193 8.23 100 5.46 76 6.86 98 194 7.73 99 5, 34 57 6.58 97 195 8.35 98 5, 25 70 6, 43 93 196 7, 63 99 5, 31 68 6, 25 92 197 7.99 98 <5 42 6, 68 96 198 8.25 98 <5 35 6, 38 91 199 7.27 92 5, 08 62 5.23(h) 5.32 (g) 62(h) 21(d) 200 7, 01 91 <5 35 <5 thirteen 201 6, 19 61 <5 24 5.51 (g) -1 (d) 202 5.57 thirty <5 eighteen <5(h) <5 (g) 38(h) 4 (g) 203 6, 02 48 <5 41 <5 (d) 14 (d) 204 6.79 88 5, 13 48 <5 (d) 13 (g) 205 8.94 99 5.65 83 7, 01 98 206 5.78 39 <5 -one <5 sixteen 207 5, 1 21 <5 2 <5 -5 208 6, 23 62 <5 thirty <5 4 209 6, 14 57 <5 25 <5 7 210 6.21 56 <5 5 <5 sixteen 211 <5 3 <5 thirteen <5 ten 212 6, 24 61 <5 7 <5 eight 213 5, 63 28 <5 eleven <5 23 214 5, 2 nine <5 nine <5 thirty 215 6.58 82 5 45 5, 13 63 216 6.36 98 <5 26 5, 35 67 217 6, 25 99 <5 37 5, 32 72 218 6.69 100 <5 51 5.65 85 219 6.86 100 <5 eighteen 5, 04 45

- 139 039788

220 6.52 54 <5 eight <5 41 220 (.HCl) 6.52 54 <5 eight <5 41 221 5, 92 45 <5 4 <5 26 222 6.55 98 <5 thirteen 5, 17 59 223 6.71 98 <5 nineteen 5.46 71 224 8.52 100 5.98 87 7, 15 99 225 7.94 99 5.75 83 7, 03 98 226 9, 06 100 6, 89 100 7, 09 100 227 8, 66 101 6, 42 95 7.34 98 228 9, 65 100 5, 92 91 7.77 100 229 6, 25 95 <5 eighteen <5 33 230 5.99 93 <5 40 5, 27 65 231 8, 87 97 5, 04 57 7.71 100 232 6, 67 99 <5 31 <5 48 233 6, 24 94 <5 ten <5 26 234 6.29 96 <5 22 5, 06 59 235 6, 91 98 <5 32 5.26 67 236 6, 68 100 <5 eighteen 5, 11 58 237 6, 73 97 <5 20 5, 09 51 238 6, 42 94 <5 35 5 48 239 6, 72 96 <5 sixteen <5 43 240 5, 81 90 <5 nine 5, 04 51 241 7, 68 97 5.76 88 6.57 94 242 6, 87 97 5.21 67 5, 09 56 243 6, 88 99 <5 thirteen 5, 44 70 244 6, 17 92 <5 12 5, 03 54 245 6, 91 99 5, 23 62 5, 66 79 246 6, 9 98 <5 25 <5 44 247 6, 81 98 5, 69 85 5, 54 77 248 6.46 95 <5 2 4.98 49 249 6, 43 95 <5 ten <5 45 250 6, 88 99 <5 28 5, 31 64 251 6.55 98 <5 17 5, 06 53

- 140039788

252 6, 15 96 <5 17 5, 17 66 253 6, 34 95 <5 17 <5 39 254 6.97 99 <5 25 5, 42 70 255 6.57 95 <5 28 5, 11 58 256 6.49 97 <5 12 5 48 257 6.58 95 <5 fifteen <5 45 258 6, 82 99 5, 81 89 5, 9 90 259 6, 37 99 5.45 82 5, 77 92 260 6.86 97 <5 23 5, 53 74 261 6, 44 95 <5 nineteen <5 45 262 6, 81 101 <5 37 5.49 74 263 6, 89 99 5, 31 65 5.29 64 264 6.98 100 4.98 47 5.48 74 265 8, 62 102 5, 2 55 7.43 98 266 6, 38 100 5, 27 74 5.58 89 267 9, 4 102 7.21 100 7.95 99 268 5, 72 82 <5 thirteen <5 39 269 8.93 99 5, 43 78 6, 25 93 270 8.34 99 <5 29 6.48 96 271 8, 66 99 5, 13 61 6.78 95 272 8, 81 97 5, 05 54 6.28 93 273 8, 63 100 6, 09 96 6, 01 96 274 6.54 97 <5 7 5, 22 63 275 8.55 100 6, 01 92 7.54 100 276 8, 65 100 5, 89 93 6.78 98 277 7, 12 99 <5 fourteen <5 27 278 9, 03 99 6, 15 97 6, 84 99 279 8, 65 99 5.55 86 6, 5 96 280 8, 6 100 6, 03 96 6, 61 99 281 8.78 100 5.65 87 6.96 99 282 8.54 100 5, 77 88 6.56 98 283 8.07 100 <5 38 6.75 98 284 8.77 101 5, 9 89 7.26 98 285 5.16 65 <5 21 <5 37

- 141 039788

286 8.16 101 <5 fourteen 5.65 79 287 8.42 100 5, 2 61 6.76 98 288 8.57 101 <5 44 6, 38 93 289 8, 62 100 6.75 98 7.2 99 290 8.23 98 5.16 53 5.85 89 291 8.57 101 5.58 86 6.29 95 292 8, 6 100 5, 33 73 6, 14 94 293 7.95 100 5.46 80 5.99 90 294 7.97 99 <5 fourteen 5, 68 81 295 9, 31 101 6, 01 94 6, 61 98 296 9, 34 99 6, 1 93 6, 92 100 297 8, 63 97 5, 23 62 6, 23 96 298 7.73 99 <5 25 <5 47 299 8, 11 99 <5 40 5, 67 84 300 8.19 100 5, 7 88 5, 54 80 301 8, 09 100 5, 2 62 5, 9 89 302 8, 19 101 5, 1 59 6.05 93 303 8, 15 99 5, 18 57 6, 27 95 304 7.4 100 5, 04 48 <5 45 305 7, 06 100 <5 7 5, 15 57 306 7.35 100 5, 35 71 5, 44 74 307 8.04 99 5, 83 88 6, 22 97 308 7.76 98 5, 34 72 5, 17 66 309 8.53 100 5, 12 53 5, 82 86 310 8, 85 100 5.41 78 6, 4 95 311 8.96 96 5, 13 62 6, 37 96 312 7, 66 99 <5 37 6.21 93 313 8, 87 97 5, 22 61 5, 91 86 314 8.24 100 5.45 73 6, 03 91 315 6.95 98 <5 20 5, 01 47 316 6.59 94 <5 5 <5 fourteen 317 7.07 99 <5 fifteen <5 34 318 5, 82 89 <5 eleven <5 thirteen 319 8.95 100 5.99 95 6.58 99

- 142 039788

320 7.27 100 <5 47 5, 15 62 321 8, 66 101 5, 09 45 6.51 97 322 9, 3 100 6, 32 95 7.73 99 323 8.41 100 6, 14 95 6.65 97 324 9, 07 100 6.58 97 7.18 100 325 8.94 100 6.51 98 7, 19 98 326 8, 67 100 6.59 97 7.22 99 327 9, 38 101 7, 11 99 7, 69 100 328 9, 2 100 5, 83 90 7.21 100 329 9.26 100 6.69 95 7, 67 100 331 9, 32 98 7.3 99 7, 84 100 332 8.32 99 5, 92 90 7.51 98 333 8.09 99 5.16 63 6, 15 93 334 8, 61 100 5, 63 84 7 98 335 9, 02 102 5, 14 52 7.06 98 336 7.73 101 5.49 78 5, 72 87 337 6.78 99 <5 43 5, 34 71 338 7, 04 99 5.26 62 5.51 70 339 8.18 101 5, 32 69 6.21 95 340 7, 15 100 5, 37 69 5.48 72 341 6.85 97 <5 33 5, 3 63 342 7.49 100 5, 35 69 5.57 77 343 7, 17 102 <5 44 5, 34 68 344 7.53 99 5, 14 61 5.59 80 345 7, 12 99 5, 08 48 5, 43 72 346 8.28 100 5, 22 68 6, 14 92 347 8.27 101 5, 14 63 6 91 348 7.47 99 5, 35 70 5, 89 92 349 7, 65 102 5, 32 64 5.75 86 350 7, 6 100 5, 31 74 5, 68 85 351 7.29 99 5, 2 69 5, 67 83 352 8.12 100 5, 74 89 6, 32 99 353 8.47 99 5, 32 69 6, 01 94 354 7, 05 100 5, 01 45 5, 35 70

- 143 039788

355 9, 03 99 6, 14 98 6, 63 98 356 8.27 99 5, 23 65 6, 03 92 357 7, 11 98 <5 34 5, 47 73 358 7.57 102 <5 40 5, 1 58 359 7.92 99 5, 4 71 5, 67 86 360 7.39 97 5, 07 fifty 5.16 55 361 8.56 100 <5 37 6.45 92 362 8.77 99 6, 06 88 6, 1 93 363 9.56 99 6.21 97 6.75 98 364 8.41 101 5, 5 75 5, 74 85 365 7.2 99 <5 24 <5 33 366 7.99 99 <5 22 5, 24 64 367 6.36 97 <5 21 <5 44 368 8.21 101 5, 19 61 6, 6 99 369 8.31 100 5, 43 72 6, 14 93 370 7.47 100 5, 84 87 6, 18 94 371 7.86 101 6.41 96 6.41 98 372 8.99 100 5, 87 90 6, 6 96 373 8.57 100 6, 27 97 6, 5 99 374 9, 13 100 6, 13 97 6, 74 98 375 8.45 101 5, 35 73 6, 09 94 376 8.37 101 5, 47 79 6.51 97 377 7.75 99 5, 01 47 5, 89 89 378 8.77 99 6, 12 96 6, 39 97 379 7.95 102 5.28 67 5.79 87 380 8.26 101 5, 4 72 6, 44 96 381 8.12 99 5 48 5, 88 88 382 7.99 99 <5 37 5, 4 75 386 8, 62 98 5, 6 79 6.26 93 387 8.54 100 5, 77 88 6.56 98 390 8, 68 98 5.59 84 7.21 99 391 8, 62 99 5.65 81 7.2 97 392 8.95 99 6, 35 97 6.58 93 394 8, 03 99 <5 27 5.65 76 396 7, 82 98 <5 56 5, 83 84 398 8.31 99 5.28 67 6, 23 95 400 7, 65 99 <5 sixteen <5 41 402 7.58 100 <5 28 5, 83 87

PDE2 Occupancy by Test Compounds

Ways.

PDE2A occupancy was assessed by ex-vivo autoradiography using [ ³ H]B-17a (described in WO 2013/000924) as the radioligand (Compound 12 in Buijnster et al., (2014). Structure-Based Design of a Potent, Selective, and Brain Penetrating PDE2 Inhibitor with Demonstrated Target Engagement.ACS Med Chem Lett.5(9):1049-53.)

Male Wistar rats (200-250 g) were treated by oral administration of vehicle

- 144039788 or increasing doses of [ ³ H]B-17a and sacrificed after 1 hour. The brain was immediately removed from the skull and subjected to rapid freezing in dry ice-cooled 2-methylbutane (-40°C). Sections of the striatum 20 µm thick were cut using a Leica CM 3050 cryostat microtome (van Hopplynus,

Belgium), placed thawed on glass slides (SuperFrost Plus Slides, LaboNord,

France) and stored at -20°C until use.

After thawing, sections were dried in cold air and incubated for 1 min with 30 nM [ ³ H]B-17a in Tris-HCl (50 mM, pH 7.4) containing 0.3% BSA. Brain sections from drug-treated and vehicle-treated animals were incubated simultaneously.

Non-specific binding was measured in sections of the cerebellum, a region of the brain that does not contain the PDE2A enzyme. After incubation, the excess [ ³ H]B-17a was washed off with ice-cold buffer 2 times for 10 min, followed by rapid immersion in distilled water. The sections were then dried in a cold air stream.

The brain sections were placed in a β-imager (Biospace, Paris) for 4 hours and the radioactivity emanating from the cut areas of the brain was quantified using the Betavision program (Biospace, Paris). Specific binding was defined as the difference between total binding in the striatum and non-specific binding in the cerebellum. The percent occupancy of the receptor by the drug administered to the animal corresponded to 100% minus the percent labeled receptor in the treated animal. To determine ED ₅₀ values, percent receptor occupancy values were plotted against dose and the equation of the most appropriate sigmoidal logarithmic dose-response curve was calculated using non-linear regression analysis using GraphPad's Prism software. ED ₅₀ values (dose of drug providing 50% receptor occupancy) at 95% confidence intervals were calculated from dose-response curves.

Tab. 6: RO=psororal; 8C=subcutaneous.

Table 6

conn. no. Employment PDE2 at 10 mg/kg Route of administration when determining PDE2 occupancy at 10 mg/kg ED ₅₀ for PDE employment Route of administration in determining ED ₅₀ For employment 1a 37 RO 2b 3 SC 2a 70 SC Behind 0 RO 4 nine RO 6 67 RO thirteen 2 RO eighteen 4 RO 22 eighteen thirty RO RO 24b 0 SC 25a 66 SC 4 RO 25b -nine RO 27 34 RO 28 49 SC 25 RO

- 145 039788

29 31 SC thirty 0 SC 32 -6 PO 33 -7 PO 35 -3 PO 36 32 PO 45.2 PO 37 -thirteen PO 38 one PO 39 -eight PO 41 eight PO 42 54 PO 43 2 PO 46 -nine PO 47 5 PO 48 96 sc 49 12 sc fifty -thirty PO 51 20 PO 52 2 PO 53 22 PO 54 39 PO 59 7 PO 60 32 PO 64 7 PO 65 -23 -nine PO PO 66 -fourteen PO 68 31 sc 69 0 PO 70 65 sc 6, 4 PO 71 49 sc 72 0 PO 73 0 PO 74 fourteen sc

- 146039788

76 17 PO 77 54 54 PO PO 5, 67 PO 78 77 SC 79 fifteen 0 PO PO 80 52 SC 8, 63 PO 82 28 PO 83 82 PO 84 45 PO 14, 13 PO 85 one SC 89 88 SC 90 73 sc 91 -7 PO 93 85 PO 7, 8 PO 95 27 sc 96 one PO 97 92 PO 99 one PO 101 43 sc 102 -4 PO 103 42 sc 106 -0 PO 107 29 PO 108 65 PO 4, 8 PO 109 43 PO 110 75 PO 8.3 PO 115 23 PO 117 eight PO 116 3 sc 120 12 PO 121 -fifteen PO 122 0 PO 123 4 PO

- 147 039788

125 -sixteen PO 126 31 PO 127 34 53 PO SC 129 21 PO 130 0 PO 131 -0 PO 132 76 SC 5 PO 133 -nineteen PO 134 21 PO 139 -eleven PO 169 3 PO 170 one PO 184 -2 PO 186 eight sc 187 0 sc 188 2 sc 189 nine PO 190 one PO 191 -eighteen PO 192 -thirteen PO 193 -eleven -3 PO sc 195 -7 -thirteen PO sc 196 0 PO 197 eight PO 220 -eight PO >80 PO 223 nineteen PO 224 -6 PO 225 -6 PO 227 eighteen sc 228 92 sc 1.5 PO 231 -thirteen sc

- 148 039788

267 59 PO eleven PO 269 22 PO 24.2 PO 270 6 PO 272 3 PO 273 65 SC 275 23 PO 277 -4 PO 278 thirty PO 281 57 PO 4.2 PO 282 47 47 PO PO 11.8 PO 283 -12 PO 284 sixteen PO 286 5 PO 287 43 PO 290 -3 PO 291 38 PO 292 23 PO 293 6 PO 294 17 PO 295 71 PO 5, 63 PO 296 88 PO 297 -2 PO 299 31 PO 300 -eleven PO 301 -7 PO 302 one PO 303 17 PO 307 eight PO 308 eighteen PO 309 12 PO 310 52 PO 9, 6 PO 311 -one PO 312 -sixteen PO

- 149 039788

313 28 RO 314 5 RO 319 37 RO 321 73 RO 3.37 RO 328 -eighteen RO 332 eight RO 334 17 RO 335 88 RO 0.985 RO 339 -one RO 346 nineteen RO 347 eleven RO 352 sixteen RO 353 fifteen RO 356 51 RO 5.55 RO 362 3 RO 363 -eleven RO 364 -5 RO 366 eighteen RO 368 eleven RO 369 nine RO 386 23 RO 387 47 47 RO RO 11, 8 RO 391 3 RO 392 46 RO 398 29 RO

Effect of Compound 110 on Synaptic Transmission

important reagents.

Sucrose preparation buffer containing (in mM) sucrose (150), NaCl (40), KC1 (4), NaH ₂ PO ₄ .H ₂ O (0.3), MgC1.6H ₂ O (7), NaHCO ₃ (26), CaCl ₂ .2H ₂ O (0.5), D-glucose (10), equilibrated with a gas mixture of 95% O ₂ and 5% CO ₂ . The artificial cerebrospinal fluid (ACSF) used during balancing and registration contained (in mM): NaCl (124), KS1 (2.7), NaH ₂ PO ₄ .H ₂ O (1.25), MgSO ₄ .7H ₂ O (1.3), NaHCO ₃ (26), CaCl ₂ .2H ₂ O (2), D-glucose (10), ascorbic acid (2), equilibrated with a gas mixture of 95% O ₂ and 5% CO ₂ . ACSF was prepared with CNQX and kynurenic acid at 50 μM and 1 mM, respectively. Test compounds were prepared immediately prior to use from a stock solution (with DMSO) in ACSF and with a final DMSO concentration that did not exceed 0.1%. All reagents were from Sigma-Aldrich unless otherwise noted.

Animals (species, weight and sex).

The animals used were male Sprague-Dawley rats weighing in the range of 145-200 g purchased from Charles River, Germany.

Obtaining sections of the hippocampus.

Horizontal brain sections (300 μm) were obtained from the mid-ventral hippocampus of male Sprague-Dawley rats anesthetized with isoflurane according to standard protocol. Sections were cut using a vibrating tissue microtome (Leica VT1200S) in cold (4° C.) sucrose preparation buffer at a speed of 0.1 mm/s. After cutting, the sections were left to equilibrate at 35°C for 20 min and then allowed to recover at rt. for at least one hour in artificial cerebrospinal fluid (ACSF). Three to four sections were obtained from one brain.

Testing system.

All data were recorded using a MEA setup commercially available from Multichannel.

- 150039788

Systems MCS GmbH (Reutlingen, Germany), consisting of a 4-channel pulse generator and a 60-channel main module with an amplifier connected to a 60-channel A/D card. The stimulation, data logging and analysis software was commercially available from Multi Channel Systems: MC Stim (II version 2.0.0) and MC Rack (version 3.8.1.0), respectively. All experiments were performed using a 3-dimensional MEA (Ayanda Biosystems, S.A., CH-1015, Lausanne, Switzerland), which consisted of 60 pointed electrodes with a height of 60 µm, the distance between which was 100 µm. MEA electrodes are made of platinum with a resistance in the range of 600-900 kOhm.

Scheme of the experiment.

The effect of test compounds on synaptic transmission was investigated by recording extracellular field potentials in sections of the hippocampus. It is well known that synaptic transmission can provide a deviation of the extracellular field potential, which reflects synchronous synaptic activity in the population of neurons surrounding the recording electrode.

Registration of extracellular field potential data.

After obtaining the brain sections were placed on the MEA chip under a microscope and 60 recording electrodes were placed in the region of the synapse of mossy fibers (dentate gyrus - CAZ) of the hippocampus. The ACSF solutions were continuously perfused at a flow rate of 2 ml/min. The temperature in the MEA chamber was maintained at 32±0.1° C. with the Peltier element positioned on the main module with the MEA amplifier. All data were recorded using an MEA setup commercially available from Multichannel Systems MCS GmbH (Reutlingen, Germany). Two adjacent electrodes in the chip were chosen to stimulate mossy fibers in the area of the chyle of the dentate gyrus, and fEPSP was recorded in the terminal zone of the SAZ area of the hippocampus. Field extracellular postsynaptic potentials (fEPSP) were evoked by stimulating the input signal on mossy fibers with two consecutive electrical pulses 30 ms apart and repeated every 60 s (pulse duration was 100 μs and stimulating current strength (μA) was 40% of the maximum amplitude ). Control experiments were performed simultaneously with sections, which were randomly assigned to be processed using a carrier medium (DMSO). N denotes the number of slices, and typically 3-4 slices were used per animal. Evoked responses at the level of postsynaptic neurons (fEPSP) were recorded if they met certain quality criteria, including: correct location, stable background (deviation within ±10% for ten consecutive minutes, amplitude >100 μV). fEPSP data from selected electrodes were collected at 5 kHz and recorded to a PC hard drive for offline analysis. Simultaneously, fEPSP amplitude values from selected electrodes were collected in real time (using the MS Rack program) to control and monitor the quality of the experiment. The data was plotted in a spreadsheet file for offline analysis.

Weak long-term potentiation (LTP) was induced with a single high-frequency stimulus (HFS) to provide fEPSP potentiation less than the maximum.

The results of this test are shown in Fig. 1 regarding the effect of Compound 110 as a PDE2 inhibitor in promoting LTP induction with a weak long term potentiation protocol.

Effect of compounds 70, 25a and 220 (free base) on synaptic transmission

important reagents.

Sucrose preparation buffer containing (in mM) NaCl (124), KCl (4.4), NaH ₂ PO ₄ .H ₂ O (1.2), MgCl.6H ₂ O (2), NaHCO ₃ (26) , CaC12.2H2O (2), D-glucose (10), equilibrated with a gas mixture of 95% O ₂ and 5% CO ₂ . The artificial cerebrospinal fluid (ACSF) used during balancing and registration contained (in mM): NaCl (124), KCl (4.4), NaH ₂ PO ₄ .H ₂ O (1.2), MgSO ₄ .7H ₂ O (2), NaHCO ₃ (26), CaCl ₂ .2H ₂ O (2), D-glucose (10), ascorbic acid (2), equilibrated with a gas mixture of 95% O2 and 5% CO2. Compounds 1, 2 and 3 were prepared immediately prior to use from a stock solution (with DMSO) in ACSF and with a final DMSO concentration that did not exceed 0.1%. All reagents were from Sigma-Aldrich unless otherwise noted.

Animals (species, weight and sex).

The animals used were male Sprague-Dawley rats weighing in the range of 145-200 g purchased from Charles River, Germany.

Obtaining sections of the hippocampus.

Rats were anesthetized with isoflurane and immediately decapitated. The brain was placed near the agarose block and cut horizontally with a blade moving from front to back. Sections were cut at a thickness of 350 µm using a vibrating tissue microtome (Leica VT1200S) in cold (4° C.) carbon dioxide-saturated artificial cerebrospinal fluid (ACSF) at a speed of 0.08 mm/s and an oscillation amplitude of 0.75 mm. After cutting, the sections were equilibrated at 35°C for 20 min and then allowed to recover at

- 151 039788 room temperature for at least one hour in ACSF. Typically, six to eight sections were obtained from each brain and three to four were used per experiment.

Testing system.

All data were recorded using the Slicemaster system, commercially available from Scientifica (UK), consisting of 4 recording stations. A platinum stimulating electrode coated with Isonel was placed in the SAZ region of the rat hippocampus. Recording microelectrodes (resistance approximately 5 MΩ) were filled with ACSF and placed in CA1 of the rat hippocampus. The location of the stimulating and recording electrodes was confirmed by applying 20 s current stimulation. A pre-registration of 20 min was used to ensure stabilization of the responses from the slices. Current stimulation was applied with an A365 current disconnector (World Precision Instruments). Data were acquired with a pClamp 10 connected to a Digidata 1440A data acquisition board (Molecular Devices, Sunnyvale, CA, USA) at a sample rate of 10 kHz with low pass filtering at 1 kHz and high pass filtering at 3 Hz. Stimuli of one hundred μs in the range of 0-100 μA were applied to elicit fEPSP and the fEPSP value was determined by measuring the maximum negative amplitude or 20-80% slope of the ascending phase. The data were analyzed offline using specially developed algorithms in IGORpro (Wavemetrics).

Scheme of the experiment.

The effect of compounds 70, 25a and 220 (free base) on synaptic transmission was investigated by recording extracellular field potentials in sections of the hippocampus. It is well known that synaptic transmission can provide a deviation of the extracellular field potential, which reflects synchronous synaptic activity in the population of neurons surrounding the recording electrode.

Registration of extracellular field potential data.

After reconstitution, sections were continuously perfused with oxygenated ACSF (2.5 ml/min). The solution was preheated in a water bath before being pumped into the recording chamber. Data registration was carried out at 32°C. We recorded simultaneously fEPSP from four independent brain slices. N denotes the number of slices, and typically 3-4 slices were used per animal. Evoked responses at the level of postsynaptic neurons (fEPSP) were recorded if they met certain quality criteria, including: correct location, stable background (deviation within ±10% for ten consecutive minutes, amplitude >100 μV). fEPSP data from selected electrodes were collected at 5 kHz and recorded to a PC hard drive for offline analysis. Simultaneously, fEPSP amplitude values were collected in real time (using Pclamp) to control and monitor the quality of the experiment. The data was plotted in a spreadsheet file for offline analysis.

I/O curves were generated and stimulation intensity was set at 50% of the range between minimum and maximum fEPSP (defined by either stimulation strength sufficient to produce a population spike or fEPSP amplitude plateau). In the case of LTP experiments, sections were then stimulated every 60 s for a 20 min base period (carrier medium with or without compound) followed by immediate theta pulse stimulation (shown in FIG. 2). After theta pulse stimulation, the sections were then stimulated every 60 s for 60 min to measure the level of LTP.

The results of this test are shown in Fig. 3 with respect to the effect of compound 70, FIG. 4 with respect to the effect of compound 25a and in FIG. 5 regarding the effect of Compound 220 (free base) in promoting LTP induction with a weak long term potentiation protocol.

PK/PD Single Dose PDE2i Study in Dogs

These studies used female and male Marshall Beagle dogs (1-6 years old): 2 males and 2 females per treatment group. Cerebrospinal fluid (CSF) was collected using a needle-guided cannula from the lateral ventricle of the brain of conscious animals connected to the machine.

CSF and blood samples at baseline were taken 2-5 days prior to dosing. Animals were fasted overnight and dosed on an empty stomach the following morning (orally by gavage). Blood and/or CSF were collected at predetermined time intervals post-dose to measure compound and cGMP levels. cGMP analysis was performed by LC-MS/MS: 25 μl of CSF was diluted with 125 μl of artificial CSF (STIL (20 ng/ml)), centrifuged and injected into a 25 μl column. The systems used were: Shimadzu SIL-30 UPLC system (Hypercarb column (50x1 mm (3 µm)), gradient: aqueous base (10 mM ammonium carbonate) acetonitrile (5% to 98% in 5.5 min) at flow rate of 250 μl/min) and an API Sciex 5500 system equipped with an ESI source (selective MRM transition (mass/charge 346.1^152.1 (hold time 75 ms)). The results of this study are summarized in Figs. 6-15.

- 152 039788

Enhancement of Synaptic Plasticity by PDE2 Inhibition in the Shaffer-CA1 Hippocampal Collateral Circuit in Anesthetized Rats: A Case Study Using Compound 110

Introduction.

Synaptic plasticity is a fundamental mechanism for many neurobiological functions. Long-term potentiation (LTP), a sustained, highly localized increase in synaptic strength in the hippocampus as well as the cerebral cortex, is the synaptic basis of memory and learning (Cooke and Bliss, Curr Opin Investig Drugs. 2005, 6(1):25-34) . The increase and decrease in the strength of a synaptic connection depends on the activity of presynaptic and postsynaptic neurons, on how the neural networks of the brain function in creating a sensory representation of many objects in memory and receiving a motor response. Various features of these synaptic changes in the intact brain are critical to the functioning of different types of neural networks and the functioning of several different neural circuits in the brain. Therefore, LTP is expected to be attenuated in age-related mental and neurodegenerative disorders such as Alzheimer's disease (Bergado and Almaguer, Neural Plast. 2002;9(4):217-32; Rowan et al., Biochem Soc Trans. 2005 ; 33: 563-7). The procedure, carried out in closely connected regions of the intact brain of animals under anesthesia, is an effective way to study persistent changes in effective density and plasticity in the hippocampal-cortex circuit after low and high frequency tetanic electrical stimulation delivered in single or double impulses (Albensi et al., Exp Neurol. 2007; 204A:1-13). Research is helping to expand our understanding of the neural circuits that underlie the development of impaired synaptic strength, i.e. to determine the direct path in the chain and the role of a specific biological target, covered by specific sites, in mediating the weakening of the synaptic connection. The procedure allows testing of pharmacological agents aimed at restoring pathological neuroplasticity, such as reversing defects in LTP and neural network density by increasing the efficiency of synaptic transmission, which is expected to have beneficial effects on associated cognitive and learning abilities (Cooke and Bliss, 2005; Albensi et al., 2007).

Phosphodiesterases (PDEs) are a class of enzymes responsible for the metabolic inactivation of the second messengers, cyclic 3',5'-adenosine monophosphate (cAMP) and cyclic 3',5'-guanosine monophosphate (cGMP) (Francis et al. Physiol Rev. 2011, 9 : 651-90). 11 PDE families were classified, inclusive, based on their structure, enzymatic activity and distribution (Omori and Kotera Circ Res. 2007; 100:309-27). The role of PDE in amplifying signal transduction via cyclic nucleotides makes these enzymes attractive targets for regulating excitability and enhancing neuronal interaction effects. In the brain, PDE2 is mainly expressed in the cerebral cortex, hippocampus, and striatum, where it controls cAMP hydrolysis. Over the past few years, research groups have focused on the development of PDE2 inhibitors that target the activity of intracellular second messengers, cGMP and cAMP, with effects on plasticity and cognitive processes (Duinen et al., Curr Pharm Des. 2015; 21:3813-28; Gomez and Breitenbucher, Bioorg Med Chem Lett. 2013; 23: 6522-7; Xu et al., Neurobiol Aging. 2015; 36:955-70; Barco et al., Expert Opin Ther Targets 2003; 7: 101-114) .

This study examined whether inhibition of PDE2 with Compound 110 results in changes in excitability or in the ability to exhibit synaptic potentiation at the synapses of the hippocampal Shaffer-CA1 collaterals in urethane-anaesthetized Sprague-Dawley rats.

Materials and methods

Animals.

These experiments were carried out in strict accordance with the guidelines of the International Association for the Certification and Accreditation of Laboratory Animal Care (AAALAC) and the Directive of the Council of the European Communities (86/609/EEC) of November 24, 1986, and were approved by the local ethical committee. Sprague-Dawley rats weighing 170-200 g at the time of surgery were housed as a group in ventilated cages with a set 12 h light/dark cycle (lights on at 07:00 am) after their arrival in the vivarium and kept under controlled environmental conditions.

Surgical intervention and electrophysiology.

Rats were anesthetized with an intraperitoneal injection of urethane at a dose of 1.5 g/kg body weight. Animals were fitted with a stereotactic electrode frame and their body temperature was continuously monitored with a rectal probe and maintained at 37°C with an electric heating pad. Additional administration of urethane (0.2-0.5 g/kg) was performed if necessary to ensure complete anesthesia. Two small holes (1 mm in diameter) were drilled in the skull at the location of the structures of the left hippocampus for stimulating and recording electrodes. Bipolar stimulating electrode; a pair of twisted wires made of tungsten (75 µm) with tips separated in the horizontal direction at a distance of 0.125 µm from each other were placed in the path of Schaffer collaterals of commisural nerve fibers (SC) (AP -3.4, ML -2.5, DV - 1.9-2.4), and

- 153 039788 a recording electrode made of tungsten was placed in the Stratum Radiatum region of the Cornu Ammonis (CA1) of the dorsal part of the hippocampus (AP -4.2, ML -4.0, DV -2.5-3.4) (Fig. 16a). The dura was pierced through both holes and the stimulating and recording electrodes were lowered very slowly (0.2 mm/min) through the cerebral cortex and the upper layers of the hippocampus into the mPP and DG of the dorsal hippocampus. Every effort was made during the surgical intervention to minimize the suffering of the animal.

The slope of the field excitatory postsynaptic potential (fEPSP) was used as a measure of excitatory synaptic transmission. Single monophasic rectangular pulses with a duration of 0.1 or 0.2 ms, generated by a DC power supply (MC, Germany), were applied, for example, to SC and evoked responses arose in CA1. Extracellular field potential signals were amplified; filtered using a filter with a bandwidth of 1 Hz to 2 kHz, digitized and analyzed using custom software. The electrodes were lowered until a negative fEPSP deflection was observed with a maximum response. To ensure stabilization of excitability, at least 30 minutes were allowed before measurements. Monophasic DC pulses were then applied at stimulus intensities ranging from 1 to 12 V to obtain input/output (I/O) curves and determine the maximum fEPSP slopes, and then the stimulus intensity that provided 50% of the maximum response (i.e., e. control pulse) was used in the following experiments.

LTP induction.

After I/O curves were determined, test stimulation was then applied every 30 seconds before and after tetanic stimulation. For each time point measured during the experiments, five recorded evoked response values were averaged at a frequency of 0.033 Hz. Baseline activity was measured every 5 minutes for at least 1 hour to ensure a stable baseline. The last 30 minutes of baseline recording (6 time points) immediately after drug administration were averaged and used as a control for LTP induction. Tetanization was induced with a 200-Hz high-frequency stimulation (HFS) protocol consisting of rectangular pulses (0.2 ms stimulus duration, 10 pulses for 20 stimuli, 2 s interval between pulses) at a stimulus intensity that caused an fEPSP slope that was about 50% of the maximum response. fEPSP was recorded for 120 min after HFS to determine possible changes in the synaptic response of SC-CA1 neurons. LTP measurements were derived from field EPSP ratios from the normalized mean slope obtained 120 min after HFS divided by the normalized mean slope collected 30 min before HFS. The slope of the estimated fEPSP was measured from the end of the stimulus-induced artifact to the minimum negative peak point. In 80% of the interval between these points, an analysis using a linear approximation using the least squares method was applied to calculate the slope of the EPSP.

Histology.

At the end of the electrophysiological study, electrical stimulation was performed with a current of 500 μA for 30 s to obtain a lesion in the area of the tips of the stimulating and recording electrodes, and the brains were collected for histological confirmation of the correct placement of the electrodes. Brain sections (20 μm) were examined using a light microscope. Animals with incorrect electrode placement were excluded from the study.

Compound 110 was dissolved in 20% cyclodextrin (CD) + 1HCl. For subcutaneous administration, compound 110 was dissolved in 20% CD+1HCl to achieve final concentrations of 2 and 4 mg/ml.

Statistical analysis.

For each animal, stable responses at baseline (before tetanus) over 30 min were averaged and the mean was normalized to 100%, and post-tetanus response data expressed relative to the mean at baseline. Comparison of vehicle and Compound 110 effects after tetanus was performed at 30 min intervals using one-way analysis of variance (ANOVA), and post hoc least significant difference (LSD) analysis was used for group comparisons.

Results.

No differences were found in the slope of the SC-CA1 fEPSP pathway among the I/O study groups, suggesting that the excitability of CA1 cells was similar in all animals (Fig. 16b, c). Basal synaptic transmission was enhanced as significant changes were found when Compound 110 (20 and 40 mg/kg) and control treated with vehicle were compared at baseline to tetanus (+8 and 9%, respectively) (Fig. 16d, e) . Within the LTP induction paradigm, subcutaneous administration of compound 110 (20 and 40 mg/kg) increased sustained (>2 h) synaptic potentiation (137 ± 8% and 142 ± 5% compared to the level for the carrier medium of 119 ± 4%, respectively). ) (Fig. 16d, inset diagram). At 0-30 min after completion of tetanization, fEPSP slopes were 153±5% and 155±4% compared to the vehicle level of 139±1%, p<0.05). At subsequent intervals of 30 min, analysis of stimulus-response curves revealed a significant sustained increase in fEPSP at a dose of 40 mg/kg (30-60 min: 146±6%, 60-90 min: 137±5% and 90-120 min: 129±5% compared with the level for the carrier medium 125±6, 116±9 and 106±13%, p<0.05, respectively). Overall, Compound 110 promotes basal synaptic transmission and LTP in vivo.

Permissible variations should not be construed as a deviation from the scope of the present invention. It will be apparent that those skilled in the art may modify the invention thus described in various ways.

CLAIM

Claims (15)

CLAIM 1. The compound characterized by the formula (I), or its stereoisomeric form, where R ^a is selected from the group consisting of H, CH ₃ , CN and CHF ₂ ; R ^B is a radical selected from the group consisting of (a) and (c) (a) , (c), where R1 is H, F or CH ₃ ; R ² is H, methyl or n-butyl; with the proviso that if R ² is H, then R 1 is F or CH ₃ ; R ³ is Ar, Het or Ar-C _2-4 alkenyl; where Ar is phenyl or naphthyl, each of which is optionally substituted with 1, 2 or 3 substituents, each independently selected from the group consisting of halogen; CN; NR ^2A R ^2B where R ^2A and R ^2B are each independently selected from H and CH ₃ ; OH; C _1-6 alkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; C _1-6 alkyl substituted with CN; C _3-6 cycloalkyl; C _1-6 alkyloxy optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; and pyrazolyl; Het represents: (i) a 5-membered heteroaryl selected from the group consisting of 1H-pyrrolyl; thienyl; furanyl; 1H-pyrazolyl; 1H-imidazolyl; 1,2-oxazolyl; 1,3-oxazolyl and thiazolyl; each of which may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halogen; C _1-4 alkyl optionally substituted with 1, 2, or 3 independently selected substituents representing a halogen atom; NR ^3A R ^3B where R ^3A and R ^3B are each independently selected from H and CH3; and furan-2-yl; or (ii) a 6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl; each of which may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halogen; OH; CN; NR ^4A R ^4B , where R ^4A and R ^4B are each independently selected from H and CH ₃ ; C _1-4 alkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; C _1-4 alkyl substituted with OH; O _3-6 cycloalkyl; C _3-6 cycloalkyloxy; C _1-4 alkyloxy; and C _1-4 alkyloxyC _1-4 alkyl; or (iii) an 8-10 membered bicyclic partially unsaturated heterocyclyl selected from the group consisting of 2,3-dihydro-1-benzofuranyl; 2H-chromenyl; 3,4-dihydro-2H-chromenyl; 2,3-dihydro-Shindolyl optionally substituted in position 1 with 1-acetyl; 2,2-difluoro-1,3-benzodioxolyl; 1,3benzodioxolyl optionally substituted with a methyl substituent; 3,4-dihydro2H-1,4-benzoxazinyl optionally substituted with C _1-4 alkyl; 5,6,7,8-tetrahydroimidazo[1,2a]pyridinyl; 5,6,7,8-tetrahydroquinolinyl optionally substituted with a halo substituent; and 2,3-dihydropyrazolo[5,1-b][1,3]oxazolyl; or (iv) a 9-10 membered bicyclic heteroaryl selected from the group consisting of 1benzofuranyl; 1-benzothiophenyl; 1H-indolyl; 1,3-benzoxazolyl; 1,3-benzothiazolyl; indolizinyl; 1H-benzimidazolyl; imidazo[1,2-a]pyridinyl; pyrazolo[1,5-a]pyridinyl; 1H-thieno[2,3c]pyrazolyl; imidazo[2,1-5]thiazolyl; pyrrolo[2,3-c]pyridinyl; thieno[3,2-5]pyridinyl; quinoline- - 155 039788 la; isoquinolinyl; quinoxalinyl; 1,8-naphthyridinyl and 1,6-naphthyridinyl; each of which may be optionally substituted with 1 or 2 substituents each independently selected from the group consisting of halogen; OH; NR ^5A R ^5B where R ^5A and R ^5B are each independently selected from H and CH3; _C1 . ₄ αalkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; and _C1 . ₄ alkyloxy; provided that the connection is not or its N-oxide, or a pharmaceutically acceptable salt. 2. Connection according to claim 1, where R ³ is Ar or Het; where Ar is phenyl optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halo; CN; OH; _C1 . ₆ αlkylα optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; _C1 . ₆ alkyl substituted with CN; _C3 . ₆ cycloalkyl and C ₁ . ₆ alkyloxy optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; Het represents: (i) a 5-membered heteroaryl selected from the group consisting of N-pyrrolyl; thienyl; furanyl; 1H-pyrazolyl; 1H-imidazolyl; 1,2-oxazolyl; 1,3-oxazolyl and thiazolyl; each of which may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halogen; _C1 . ₄ αalkyl optionally substituted with 1, 2, or 3 independently selected substituents representing a halogen atom; NR ^3a R ^3b , where R ^3a and R ^3b are each independently selected from H and CH ₃ ; and furan-2-yl; or (ii) a 6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl; each of which may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halogen; OH; CN; NR ^4a R ^4b where R ^4a and R ^4b are each independently selected from H and CH3; _C1 . ₄ alkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; _C3 . ₆ cycloalkyl; _C3 . ₆ cycloalkyloxy and C ₁ . ₄ αalkyloxy; or (iii) an 8-10 membered bicyclic partially unsaturated heterocyclyl selected from the group consisting of 2,3-dihydro-1-benzofuranyl; 2H-chromenyl; 3,4-dihydro-2H-chromenyl; 2,3-dihydro-Shindolyl optionally substituted in position 1 with 1-acetyl; 2,2,-difluoro-1,3-benzodioxolyl; 1,3benzodioxolyl optionally substituted with a methyl substituent; 5,6,7,8 tetrahydroimidazo[1,2-a]pyridinyl; 5,6,7,8-tetrahydroquinolinyl optionally substituted with a halogen substituent; and 2,3-dihydropyrazolo[5,1-b][1,3]oxazolyl; or (iv) a 9-10 membered bicyclic heteroaryl selected from the group consisting of 1benzofuranyl; 1-benzothiophenyl; 1H-indolyl; 1,3-benzoxazolyl; 1,3-benzothiazolyl; indolizinyl; 1H-benzimidazolyl; imidazo[1,2-a]pyridinyl; pyrazolo[1,5-a]pyridinyl; 1H-thieno[2,3c]pyrazolyl; thieno[3,2-b]pyridinyl; quinolinyl; 1,8-naphthyridinyl and 1,6-naphthyridinyl; each of which may be optionally substituted with 1 or 2 substituents each independently selected from the group consisting of halogen; OH; NR ^5a R ^5b where R ^5a and R ^5b are each independently selected from H and CH3; _C1 . ₄ αalkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; and _C1 . ₄ alkyloxy; or a pharmaceutically acceptable salt thereof. 3. A compound according to claim 1 or 2, wherein R ^A is CH3 or CHF ₂ . 4. A compound according to any one of claims 1 to 3, wherein R ³ is Het. 5. A compound according to any one of claims 1 to 4, wherein R ³ is: (i) a 6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl; each of which may be optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of halogen; OH; CN; NR ^4a R ^4b where R ^4a and R ^4b are each independently selected from H and CH3; _C1 . ₄ alkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; _C3 . ₆ cycloalkyl; - 156 039788 _C3 . ₆ cycloalkyloxy and C _1-4 alkyloxy; or (ii) a 9-10 membered bicyclic heteroaryl selected from the group consisting of 1benzofuranyl; 1-benzothiophenyl; 1H-indolyl; 1,3-benzoxazolyl; 1,3-benzothiazolyl; indolizinyl; 1H-benzimidazolyl; imidazo[1,2-a]pyridinyl; pyrazolo[1,5-a]pyridinyl; 1N-thieno[2,3c]pyrazolyl; thieno[3,2-b]pyridinyl; quinolinyl; 1,8-naphthyridinyl and 1,6-naphthyridinyl; each of which may be optionally substituted with 1 or 2 substituents each independently selected from the group consisting of halogen; OH; NR ^5a R ^5b , where R ^5a and R ^5b are each independently selected from H and CH ₃ ; _C1 . ₄ alkyl optionally substituted with 1, 2 or 3 independently selected substituents representing a halogen atom; and _C1 . ₄ alkyloxy. 6. The compound according to any one of claims 1 to 5, characterized by the formula (I-a) or (I-c), (Ι-a), (1-c) where R ^a , R ² and R ³ are defined in any one of claims 1-5. 7. A compound according to any one of claims 1 to 6, wherein the compound is 8. A pharmaceutical composition containing a therapeutically effective amount of a compound according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier. 9. The use of a compound according to any one of claims 1 to 7 as a medicament for the treatment or prevention of a disorder of the central nervous system. 10. The use of a compound according to any one of claims 1 to 7 for the treatment or prevention of a central nervous system disorder selected from Alzheimer's disease; stroke learning disabilities; a disorder or condition including, as a symptom, attention deficit disorder and/or cognitive impairment; mild cognitive impairment; age-related cognitive impairment; cognitive impairment associated with perception, attention, learning or memory; and memory disorders. 11. Use according to claim 10 wherein the disorder of the central nervous system is Alzheimer's disease. 12. A process for the preparation of a pharmaceutical composition according to claim 8, characterized in that the pharmaceutically acceptable carrier is intimately mixed with a therapeutically effective amount of a compound according to claims 1 to 7. 13. The use of a compound according to any one of claims 1 to 7 in combination with an additional pharmaceutical agent for the treatment or prevention of a condition listed in any of claims 10, 11. 14. Product containing: (a) a compound according to any one of paragraphs. 1-7 and (b) an additional pharmaceutical agent in the form of a combined preparation for simultaneous, separate or sequential use in the treatment or prevention of the condition listed in any of claims 10, 11. 15. A method of treating a disorder selected from Alzheimer's disease; stroke learning disabilities; a disorder or condition including, as a symptom, attention deficit disorder and/or cognitive impairment; mild cognitive impairment; age-related cognitive impairment; cognitive impairment associated with perception, attention, learning or memory; and memory disorders, comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of claims 1-7.